Team:GeorgiaTech/WeekSeven

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9/13/2010

Christina, Christian, Scott, Debika, Margo

Started from new aliquots of primers, 1:10 dilution of primer stock in MilliQ H2O. Changes are bolded.

Reactions: HybB F+R, OmpA F+R, AOX1A F&R, and AOX1B F&R.

New PCR protocol:

26.5 uL H2O

10 uL TaQ 5X Reaction buffer

5 uL forward primer

5 uL reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

2 uL template DNA

0.5 uL polymerase enzyme, TaQ

Total Volume= 50 uL

NOTE: We multiplied the entire protocol by 2 to get 50 uL total volume for this attempt

We are reducing number of experiments at once (e.g. not all setups at once, just a few) - HybB, OmpA, AOX1A F&R, and AOX1B F&R.

We are preparing two strips for PCR using this recipe and setup. We are then running them side-by-side in the PCR machine, one strip using the old cycle program, and one strip with two key modifications (suggested by Megan). We increased number of PCR cycles from 29 to 34 cycles, and reduced annealing temperature to 52 degrees. (If we go to low with the annealing temperature, we will be able to tell because we will see lots of bands on PCR.)

Making gel for PCR

1. Add 0.35g agarose to 35mL autoclaved water.

2. Add 3.5mL 1X TBE

3. Heat in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don’t vaporize it, especially near your face!

4. Add 38.5սL EtBr (edited from 45 uL, make 1000X)

5. Pour gel and allow to harden.

Goals: Purify the PCR reactions and look at them on a gel

See protocols page for PCR purification

note: unless we want to keep the DNA for future use (unless we NEED pure DNA), the purification step is not necessary. We could have just run the DNA without the purification step... EtBr is an intercalator that will only bind the DNA anyway.

(Pics from 9/11 and 9/13 were taped to the lab bench for group reference -- maybe get Megan and/or Richard to help us interpret.)

Starter cultures for cryostocks

Made starter cultures (3uL CARB + 3mL LB + cells) from triple smear plates (9/10/2010), and put in the incubator for 24 hours.. Tomorrow (on 9/14/2010), make cryostocks from these starter cultures. Labelled according to insert.

9/14/2010

Results from 9/13/2010: The previous gel had fairly good AOX bands but after consulting with Richard we decided that the other samples weren’t represented in the gel. Further, we should have seen primer bands near the end, so in future gels it’s important not to let samples run off.

Miniprep

In order to prepare for another gel, a miniprep of the following samples from the starter cultures made on 9/13/2010 were run:

1. PSB1A3

2. AOX1a

3. ompA

4. hybB

5. AOX1b

The contents were labeled and stored in the -20 freezer for further use in the gel.

Crystocks

Cryostocks were made from starter cultures grown on 9/13/2010. Two distinct colonies were taken from each plate - so there are duplicates of each. Stored in the -80C labelled:

9-14 clg HybB (2)

9-14 clg ompA (2)

9-14 clg AOX1a (2)

9-14 clg AOX1b (2)

9-14 clg psb1A3 (2) [note: 1 of these starter cultures turned red, the other did not.]

9/15/2010

Goals: Perform PCR on the new minipreps from 9/14/2010 (using new 1:10 aliquots of primers from 9/13/2010)

PCR Protocol

26.5 uL H2O

10 uL TaQ 5X Reaction buffer

5 uL forward primer

5 uL reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

2 uL template DNA

0.5 uL polymerase enzyme, TaQ

Total Volume= 50 uL

Notes: Wear gloves while doing the reaction. Keep all reagents on ice, including the PCR reactions. Add in the order of the protocol- get out the enzyme and place on ice right before you are about to use.

Reactions Done in PCR:

HyBb F,R
OmpA F,R
Aox1a F,R
Aox1a F,R2
Aox1b F,R
Aox1b F,R2

Notes: We are using a master mix of Water, TAQ Buffer, DNTPS, and TAQ. Add this to all the tubes (everything on ice), then add all the DNA reagents.

Results from nanospec:

Sample Concentration (ng/uL)

hybB 155.2

ompA 70.2

Aox1a 241.3

Aox1b 253.2

psb1A3 53.6

Making gel for PCR

1. Add 0.35g agarose to 36 mL 1 x TBEautoclaved water.

2. Heat in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don’t vaporize it, especially near your face!

3. Add 35 սL EtBr (edited from 45 uL, make 1000X)

4. Pour gel and allow to harden.

9/16/2010

Goals:

Interpret gel results of the PCR from 9/15/2010
Perform PCR (from 9/15/2010) again, this time with:

PHUSION Polymerase/Buffer
RFP

What we did:

(Christina, Rob)

Performed PCR with Phusion
Reactions:

HyBb F,R
OmpA F,R
Aox1a F,R
Aox1a F,R2
Aox1b F,R
Aox1b F,R2
RFP F,R
RFP F2,R

Performed miniprep of pSB1A3 from cell culture grown on 9/15/2010 (non-red)

PCR Protocol

26.5 uL H2O

10 uL PHUSION 5X Reaction buffer

5 uL forward primer

5 uL reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

2 uL template DNA

0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

Miniprep of pSB1A3

1. Remove inoculation tubes from inoculation (37C shaker).

2. Obtain P1 buffer from 4C refrigerator.

3. Take centrifuge tubes and add 1.5mL of inoculated cells.

4. Centrifuge at 3000 rpm (low) for 1-2 min.

5. Spin until white pellet of cells forms at the bottom and liquid is more clear.

6. Take off supernatant and discard.

7. Repeat steps 4-6.

8. Resuspend pelleted bacterial cells in 250սL P1 buffer.

9. Add 250սL P2 buffer and invert 4-6 times (DO NOT VORTEX - doing so will shear DNA!)

10. Add 350սL buffer N3 and immediately invert 4-6 times.

11. Centrifuge for 10 min. at 13,000 rpm.

12. Take supernatant and add to spin columns.

13. Spin 30-60 sec. and discard flow through.

14. Wash column with 750սL buffer PE and centrifuge 1 min.

15. Discard flow through and centrifuge and additional minute.

16. Please column into a clean 1.5mL microcentrifuge tube.

17. Elute DNA by adding 30սL dH2O.

18. Let stand for 1 min., then centrifuge for 1 min.

Cryostock of hybB cells and pSB1A3 (from cultures on 9/15/2010)

900 uL of cells

100 uL DMSO

Total 1 mL

Mix, place in -80c Freezer

For next time

Confirm all the predicted sizes match up with the observed sizes on the gel of the PCR from 9/15/2010
If all are confirmed (Christina has confirmed, Christian and Scott confirmed hybb, ompa), then proceed with PCR purifications of 40 uL of each reaction, then RE digests, then ligations to construct each construct

9/17/2010

Goals:

Since the PCR was run on 9/16 using Phusion (high fidelity), we need to:
A) Run gel (2 uL each)

B) PCR purify (leftovers from the 50 uL stock= 48 uL)

C) Nanospec

D) Digest with restriction enzymes (runs overnight)

Protocols

Make a gel for running PCR products from 9/16/2010 to make sure the PCR was successful.

Making gel for PCR (1% agarose gels):

Added 180 mL 1x TBE and 1.8g agarose

Heated up until agarose was no longer visible

Let cool (approx. 10 min) until there are NO MORE VAPORS

Added 180 սL Ethidium bromide (1000X)

Pour into gels and allow to harden (large wells hold about 35 mL).

Note: it is ok to make 6 gels at once according to the recipe above and store them. It is not necessary to “immediately” use gels as long as they are stored properly. From now on, when you make 1% gels, just fill all 6 wells at once.

Gel of the PCR from 9/16/2010 was run today and imaged:

volumes:

loaded 4 ul of the aox reactions, 7 ul of ompa and hybB, and 5 ul of the RFP reactions

See protocols page for PCR purification

Nanospec results (PCR from 9/16/2010)

AOX1a-F,R = 99.3 ng/uL

AOX1a-F,R2 = 50.0 ng/uL

AOX1b-F,R = 48.5 ng/uL

AOX1b-F,R2 = 42.1 ng/uL

OmpA-F,R = 27.3 ng/uL

RFP-F2,R = 10.8 ng/uL

Hyb-F,R = 26.2 ng/uL

RFP-F,R = 8.8 ng/uL

Restriction Enzyme Digest General Process

- check buffers (NEB site)

-check if BSA required (NEB site)

- Start w/ HybB and RFP (start @ step A) / pBS1A3 (start @ step D)

Recipe for : 50 uL RXN

DNA (1-2 ug- based on nanospec results)

1 uL each enzyme

5 uL 10X buffer

1 uL BSA(if needed)

Today we are digesting just the HybB-F,R and RFP-F,R in order to do the ligation on this simple construct. The rest of the PCR-ed, purified, and nanoscpec’ed building blocks went into the freezer for later use.

RE Digest Recipe for HybB

40 uL HybB-F,R (based on the nanospec results above)

1 uL EcoRI

1 uL NotI

4.7 uL 10X buffer for EcoRI [Edit 9/17/2010. Only EcoRIbuffer)

1 uL 10x BSA (based on this specfic RE mix-- BSA is required)

Place into an eppe tube, pipet gently to mix. Incubate at 37 degrees in water bath overnight.

RE Digest Recipe for RFP-F,Rr

40 uL RFP-F,R (based on the nanospec results above) [Edit 9/17/2010, 110 ul not there, reduced to 40]

1 uL SpeI (edited changed from notI to speI )

1 uL NotI

5 uL 10X buffer for EcoRI [Edit, only EcoRI buffer]

1 uL10x BSA (based on this specfic RE mix-- BSA is required)

Place into an eppendorf tube, pipette gently to mix. Incubate at 37 degrees in water bath overnight.

Results

Gel of multiple PCR reactions

For Future

We have a strategy for each construct, written on the board. We must digest each PCR product to build our building blocks, then construct each construct through a series of ligations, digest, PCRs etc (See 9.18.2010 for strategy)

9/18/2010

Goals

PCR purify the hybB F,R and RFP F,R (rxns from 9/16/2010)

Notes:

RFP F,R was accidently digested with NotI and not SpeI.
What to do? We will need to add 1 uL of SpeI and run overnight, so that the SpeI site is cleaved.
Just in case we need this RFP, we PCR-purified it today and stored in freezer, clearly labeled PARTLY DIGESTED RFP. If we need to use this RFP, it will need to be digested by SpeI.
RFP band on the gel pic from 9/17/2010 was faint and nanospec showed low conc (8.8 ng /uL) versus the higher yields of the other rxns.

Ran PCR purification on HybB, stored in freezer. This HybB was nanospec’ed on Friday and had a concentration of 26.2 ng/uL. This has been digested by the right RE’s, EcoRI and NotI, and ready to be ligated with RFP when it has been appropriately digested.

See protocols page for PCR purification

BASIC PLAN FOR BUILDING PLASMID CONSTRUCTS

from the primered building blocks, which are stored in our freezer

A) RUN GEL - 2 uL each - to check size - this step completed for all building blocks

B) PCR PURIFY leftovers - this step completed for all building blocks

C) NANOSPEC - record concentrations - this step completed for all building blocks

D) DIGEST with restriction enzymes, overnight

E) PCR PURIFY digestion products

F) LIGATION of gene building blocks - we are starting with HybB & RFP

G) PCR ligation products, HybB-RFP

H) DIGEST products, HybB-RFP

I) RUN GEL on small amount of digestion products, HybB-RFP

J) PCR PURIFY the rest of the digestion products, HybB-RFP

K) DIGEST the vector - we are using pBS1A3

L) PCR PURIFY the vector

M) LIGATION of gene with vector, HybB-RFP and pBS1A3

N) TRANSFORMATION of plasmid into E. coli, run overnight

Note- Do a nanospec after PCR purifications

NOTES:

Steps in BOLD are going to be repeated with each plasmid construct. Steps A,B, and C have already been done for each building block and won’t be repeated unless there is a specific issue with a particular building block.
Steps K and L do not necessarily need to wait until step J is completed to be run. They are listed this way so there is no confusion about what is being digested or PCR purified at each step.

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Re-suspended pellet in 250 &#1405;L PI buffer</span></p><p class="c1"><span class="c0">2. Added 250 &#1405;L P2, inverted about 5 times</span></p><p class="c1"><span class="c0">3. Added 350 &#1405;L N3, mix immediately, inverted 4-6 times</span></p><p class="c1"><span class="c0">4. Centrifuged 10 min, 13k RPM</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Enzyme digest</span></p><p class="c1"><span class="c0">5. Poured supernatant into spin column</span></p><p class="c1"><span class="c0">6. Centrifuged 30-60 seconds and discarded flow-through</span></p><p class="c1"><span class="c0">7. Washed column with 750 &#1405;L PE and centrifuged 30-60 seconds</span></p><p class="c1"><span class="c0">8. Discarded flowthrough, centrifuged 1 min</span></p><p class="c1"><span class="c0">9. Eluted by placing column in new 1.5 mL tube, added 30 &#1405;L EB. Let stand 5 min.</span></p><p class="c1"><span class="c0">10. Centrifuged 5 minutes, 5k RPM</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/6/2010</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Prepared 1% agarose gels:</span></p><p class="c1"><span class="c0">Added 180 mL 1x TBE and 1.8g agarose</span></p><p class="c1"><span class="c0">Heated up until agarose was no longer visible</span></p><p class="c1"><span class="c0">Let cool (approx. 10 min)</span></p><p class="c1"><span class="c0">Added 180 &#1405;L Ethidium bromide (1000X)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR purifcation of the cell cultures from 8/5/2010</span></p><p class="c1"><span class="c0">1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene.</span></p><p class="c1"><span class="c0">2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).</span></p><p class="c1"><span class="c0">3. Placed a spin column in a provided 2 mL collection tube.</span></p><p class="c1"><span class="c0">4. To bind DNA, applied the sample to the column and centrifuge at 17,900g for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">5. Discarded flow-through. Placed the column back in the same tube.</span></p><p class="c1"><span class="c0">6. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">7. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.</span></p><p class="c1"><span class="c0">8. Placed the column in a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">9. To elute DNA, added 50 &micro;L Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0 &ndash; 8.5) to the center of the membrane and centrifuged the column for 1 min. Alternatively, for increased DNA concentration, added 30 &nbsp;&micro;L elution buffer to the center of the membrane, let the column sit for 1 min, and then centrifuged.</span></p><p class="c1"><span class="c0">10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Gel Extraction Protocol</span></p><p class="c1"><span class="c0">1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.</span></p><p class="c1"><span class="c0">2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 &mu;L).</span></p><p class="c1"><span class="c0">3. Incubated at 50&ordm;C for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 &ndash; 3 min during the incubation.</span></p><p class="c1"><span class="c0">4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).</span></p><p class="c1"><span class="c0">5. Added 1 gel volume of isopropanol to the sample and mixed.</span></p><p class="c1"><span class="c0">6. Placed a QIAquick spin column in a provided 2 mL collection tube.</span></p><p class="c1"><span class="c0">7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.</span></p><p class="c1"><span class="c0">8. Discarded flow-through and placed QIAquick column back in the same collection tube.</span></p><p class="c1"><span class="c0">9. Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.</span></p><p class="c1"><span class="c0">10. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.</span></p><p class="c1"><span class="c0">11. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).</span></p><p class="c1"><span class="c0">12. Placed QIAQuick column into a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">13. To elute DNA, added 50 &nbsp;&mu;L of Buffer EB (1 mM Tris-Cl, pH 8.5) or water (pH 7.0 &ndash; 8.5) to the center of the QIAquick membrane and centrifuged the column for 1 min. Alternatively, for increased DNA concentration, added 30 &nbsp;&mu;L elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuged for 1 min.</span></p><p class="c1"><span class="c0">14. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/9/2010</span></p><p class="c1"><span class="c0 c2">Ligation</span></p><p class="c1"><span class="c0 c7">Purpose: Insert mRFP into pCold vector</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0">In a microcentrifuge tube add:</span></p><p class="c1"><span class="c0">1. vector DNA (pCold)</span></p><p class="c1"><span class="c0">2. insert DNA (mRFP)</span></p><p class="c1"><span class="c0">3. Ligase 10X buffer</span></p><p class="c1"><span class="c0">4. T4 DNA Ligase </span></p><p class="c1"><span class="c0">5. Nuclease-free water to a final volume of 10&#1405;L. </span></p><p class="c1"><span class="c0">6. Incubate reaction at 4C overnight.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">*(pCold: 5700 bp; mRFP: 700 bp)</span></p><table cellpadding="0" cellspacing="0" class="c19"><tbody><tr><td class="c6"><p class="c8"><span class="c0">Vector:insert</span></p></td><td class="c25"><p class="c8"><span class="c0">1:1</span></p></td><td class="c21"><p class="c8"><span class="c0">1:2</span></p></td><td class="c21"><p class="c8"><span class="c0">1:4</span></p></td><td class="c21"><p class="c8"><span class="c0">1:8</span></p></td></tr><tr><td class="c6"><p class="c8"><span class="c0">pCold (&#1405;L)</span></p></td><td class="c25"><p class="c8"><span class="c0">2</span></p></td><td class="c21"><p class="c8"><span class="c0">2</span></p></td><td class="c21"><p class="c8"><span class="c0">2</span></p></td><td class="c21"><p class="c8"><span class="c0">4</span></p></td></tr><tr><td class="c6"><p class="c8"><span class="c0">mRFP (&#1405;L)</span></p></td><td class="c25"><p class="c8"><span class="c0">3</span></p></td><td class="c21"><p class="c8"><span class="c0">4.7</span></p></td><td class="c21"><p class="c8"><span class="c0">3</span></p></td><td class="c21"><p class="c8"><span class="c0">3</span></p></td></tr><tr><td class="c6"><p class="c8"><span class="c0">10X buffer (&#1405;L)</span></p></td><td class="c25"><p class="c8"><span class="c0">1</span></p></td><td class="c21"><p class="c8"><span class="c0">1</span></p></td><td class="c21"><p class="c8"><span class="c0">1</span></p></td><td class="c21"><p class="c8"><span class="c0">1</span></p></td></tr><tr><td class="c6"><p class="c8"><span class="c0">T4 Ligase (&#1405;L)</span></p></td><td class="c25"><p class="c8"><span class="c0">0.5</span></p></td><td class="c21"><p class="c8"><span class="c0">0.5</span></p></td><td class="c21"><p class="c8"><span class="c0">0.5</span></p></td><td class="c21"><p class="c8"><span class="c0">0.5</span></p></td></tr><tr><td class="c6"><p class="c8"><span class="c0">dH2O (&#1405;L)</span></p></td><td class="c25"><p class="c8"><span class="c0">3.5</span></p></td><td class="c21"><p class="c8"><span class="c0">1.8</span></p></td><td class="c21"><p class="c8"><span class="c0">3.5</span></p></td><td class="c21"><p class="c8"><span class="c0">1.5</span></p></td></tr><tr><td class="c6"><p class="c8"><span class="c0">total (&#1405;L)</span></p></td><td class="c25"><p class="c8"><span class="c0">10</span></p></td><td class="c21"><p class="c8"><span class="c0">10</span></p></td><td class="c21"><p class="c8"><span class="c0">10</span></p></td><td class="c21"><p class="c8"><span class="c0">10</span></p></td></tr></tbody></table><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Making LB media</span></p><p class="c1"><span class="c0">To make 1L LB (Lysogeny broth) medium: </span></p><p class="c1"><span class="c0">5g Yeast abstract</span></p><p class="c1"><span class="c0">10g Tryptone</span></p><p class="c1"><span class="c0">5g NaCl</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">To make solid plates, follow the above recipe and add 16g agar per 1L.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Prepared 2L of LB media, 1L liquid broth and 1L to make plates (don&rsquo;t fill 1L bottles full - make 4 bottles 500 mL each). &nbsp;</span></p><p class="c1"><span class="c0">Autoclave, leaving lids slightly loosened.</span></p><p class="c1"><span class="c0">Let cool in water bath at 55C.</span></p><p class="c1"><span class="c0">Add antibiotic to LB media used for plates.</span></p><p class="c1"><span class="c0">For the agar plates, pipetted 25mL into each plate</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Incubating reactions</span></p><p class="c1"><span class="c0">The bacteria were left at 4C overnight.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/10/2010</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Heat shock transformation of the plasmids into our bacteria</span></p><p class="c1"><span class="c0">10 &#1405;L Nova Blue cells + 5 &#1405;L of ligation rxn</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. Left cells and ligation reaction products on ice.</span></p><p class="c1"><span class="c0">2. Added plasmid (5&#1405;L) (ligation product) to cells. Mix gently by swirling pipet tip in mixutre (DO NOT ASPIRATE).</span></p><p class="c1"><span class="c0">3. Left cells on ice for 10-30 min.</span></p><p class="c1"><span class="c0">4. Applied heat shock of 45 seconds in 42C bath.</span></p><p class="c1"><span class="c0">5. Put tubes on ice for 2 min. </span></p><p class="c1"><span class="c0">6. Added 250 &#1405;L of LB (room temp.)</span></p><p class="c1"><span class="c0">7. Incubated 1 hour at 75C (9/1 Edit: Seems improbably high- probably 37 C)</span></p><p class="c1"><span class="c0">8. Plated 100&#1405;L, then remaining amount on separate LB/CARB plates.</span></p><p class="c1"><span class="c0">9. Incubated overnight at 37C.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/11/2010</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">Inoculation</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. Add 5mL LB and 5&#1405;L CARB into test tubes (16 total) </span></p><p class="c1"><span class="c0">2. Pick 2 well-defined colonies from each plate (labelled sequentially). </span></p><p class="c1"><span class="c0">3. With an inoculation loop, get 1 colony from transformation plate and place into test tube.</span></p><p class="c1"><span class="c0">4. Repeat for each colony (16 total).</span></p><p class="c1"><span class="c0">5. Inoculate overnight at 37C.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/12/2010</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">Miniprep</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. Remove inoculation tubes from inoculation (37C shaker).</span></p><p class="c1"><span class="c0">2. Obtain P1 buffer from 4C refrigerator.</span></p><p class="c1"><span class="c0">3. Take centrifuge tubes and add 1.5mL of inoculated cells. </span></p><p class="c1"><span class="c0">4. Centrifuge at 3000 rpm (low) for 1-2 min. </span></p><p class="c1"><span class="c0">5. Spin until white pellet of cells forms at the bottom and liquid is more clear. </span></p><p class="c1"><span class="c0">7. Repeat steps 4-6. </span></p><p class="c1"><span class="c0">8. Resuspend pelleted bacterial cells in 250&#1405;L P1 buffer. </span></p><p class="c1"><span class="c0">9. &nbsp;Add 250&#1405;L P2 buffer and invert 4-6 times (DO NOT VORTEX - doing so will shear DNA!)</span></p><p class="c1"><span class="c0">10. Add 350&#1405;L buffer N3 and immediately invert 4-6 times. </span></p><p class="c1"><span class="c0">11. Centrifuge for 10 min. at 13,000 rpm. </span></p><p class="c1"><span class="c0">12. Take supernatant and add to spin columns. </span></p><p class="c1"><span class="c0">13. Spin 30-60 sec. and discard flow through. </span></p><p class="c1"><span class="c0">14. Wash column with 750&#1405;L buffer PE and centrifuge 1 min. </span></p><p class="c1"><span class="c0">15. Discard flow through and centrifuge an additional minute.</span></p><p class="c1"><span class="c0">16. Please column into a clean 1.5mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">17. Elute DNA by adding 30&#1405;L dH2O. </span></p><p class="c1"><span class="c0">18. Let stand for 1 min., then centrifuge for 1 min. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Double digest</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Into an Eppendorf tube add:</span></p><p class="c1"><span class="c0">1. ~ 25&#1405;L DNA</span></p><p class="c1"><span class="c0">2. 10&#1405;L autoclaved H2O.</span></p><p class="c1"><span class="c0">3. 4&#1405;L 10X buffer.</span></p><p class="c1"><span class="c0">4. 0.5&#1405;L Nde1 enzyme.</span></p><p class="c1"><span class="c0">5. 0.5&#1405;L Xho1 enzyme.</span></p><p class="c1"><span class="c0"> </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/13/2010</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">Making gel for PCR</span></p><p class="c1"><span class="c0">1. Add 0.5g agar to 50mL autoclaved water.</span></p><p class="c1"><span class="c0">2. Add 5mL 1X TBE</span></p><p class="c1"><span class="c0">3. &nbsp;Allow to cool. </span></p><p class="c1"><span class="c0">4. Add 45&#1405;L EtBr </span></p><p class="c1"><span class="c0">5. Pour gel and allow to harden. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Running PCR</span></p><p class="c1"><span class="c0">1. Fill gel to top with 1X TBE. </span></p><p class="c1"><span class="c0">2. &nbsp;Take 3&#1405;L loading dye and make a spot on parafilm. </span></p><p class="c1"><span class="c0">3. &nbsp;Add 10&#1405;L DNA to dye on parafilm. &nbsp;Pipette up and down to mix (NO BUBBLES). </span></p><p class="c1"><span class="c0">4. &nbsp;Load 5&#1405;L of ladder at each end. </span></p><p class="c1"><span class="c0">5. &nbsp;Add DNA + dye to wells (total 13&#1405;L).</span></p><p class="c1"><span class="c0">6. &nbsp;Put lid on. Run at 100V, until about 1/2 way down the plate.</span></p><p class="c1"><span class="c0">7. &nbsp;Transport in tubberware to image. </span></p><p class="c1"><span class="c0">8. &nbsp;Lanes that appeared to &ldquo;run&rdquo; well after imaging were inoculated on 8/16/2010.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/16/2010</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">Inoculation of cell cultures 5, 8 and 9</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. In a Eppendorf tube add 3&#1405;L carbinicillin (CARB) to 3 mL LB media. </span></p><p class="c1"><span class="c0">2. Inoculate at 37C overnight.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/17/2010</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Cell culture tubes 5, 8, and 9 from 8/16/2010 were removed from inoculation and used for two purposes:</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Freezing a portion of inoculated cells for future use</span></p><p class="c1"><span class="c0">300 &#1405;L of glycerol were added to 200 &#1405;L of each culture and tapped gently until the culture had mixed with the glycerol. These were then marked and stored in the freezer in the upstairs lab.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Addition of IPTG to induce expression of pCOLD</span></p><p class="c1"><span class="c0">To test the effects of IPTG on mRFP (and thus pCOLD) expression, we prepared 4 concentrations of IPTG for each of the 3 tubes of bacteria (5, 8, and 9), resulting in 12 tubes. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Each contained:</span></p><p class="c1"><span class="c0">930&#1405;L LB media</span></p><p class="c1"><span class="c0">50&#1405;L culture</span></p><p class="c1"><span class="c0">10&#1405;L CARB</span></p><p class="c1"><span class="c0">10&#1405;L IPTG</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">where the 10&#1405;L of IPTG were calculated serial dilutions insured to give an overall IPTG concentration of 0.1 M (10^-3), 1 mM (10^-5), 10 &#1405;M (10^-7), and 100 nM (10^-9) for each tube number 5, 8, and 9. The resulting 12 samples were put in the incubator at 37C overnight. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">If everything goes according to plan, it should be possible to see some RFP expression tomorrow.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/18/2010</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Expression of mRFP should have been detectable by red coloring in the samples after incubation. &nbsp;Nothing was seen, and as such the samples were discarded. WE PUT THEM IN THE WRONG CELLS! Novablue cells are inapropriate for this type of transformation... redo the transformation with BL21 cells.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/19/2010</span></p><p class="c1"><span class="c0 c2">Transformation into BL21 cells</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Adding IPTG to plates:</span></p><p class="c1"><span class="c0">1. &nbsp;Spread 50&#1405;L IPTG on 2 LB/carb plates (50&#1405;L each plate).</span></p><p class="c1"><span class="c0">2. Wait 1 hour to allow IPTG to soak into media.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Preparing cells: </span></p><p class="c1"><span class="c0">3. Chill 2 empty 1.5&#1405;L Eppendorf tubes, 1:4 ligation reaction product, and 20&#1405;L BL21 cells on ice.</span></p><p class="c1"><span class="c0">4. Aliquot 10&#1405;L of BL21 cells into Eppendorf tubes (10&#1405;L each tube).</span></p><p class="c1"><span class="c0">5. In each tube add 1&#1405;L 1:4 ligation reaction mixture. </span></p><p class="c1"><span class="c0">6. Allow to sit on ice for 10-30 min.</span></p><p class="c1"><span class="c0">7. &nbsp;Place in 42C bath for 30-45 sec. </span></p><p class="c1"><span class="c0">8. Immediately place back on ice for 2 min. </span></p><p class="c1"><span class="c0">9. Add 250&#1405;L room temp. LB media to reaction tube.</span></p><p class="c1"><span class="c0">10. &nbsp;Places tubes in 37C incubator/shaker for 1 hr.</span></p><p class="c1"><span class="c0">11. Spread 125&#1405;L of incubated cells onto labelled LB/CARB plates according to below:</span></p><p class="c1"><span class="c0">&nbsp;</span></p><table cellpadding="0" cellspacing="0" class="c19"><tbody><tr><td class="c15"><p class="c8"><span class="c0">Plate </span></p></td><td class="c15"><p class="c8"><span class="c0">IPTG</span></p></td><td class="c15"><p class="c8"><span class="c0">CELLS</span></p></td><td class="c15"><p class="c8"><span class="c0">TEMP</span></p></td></tr><tr><td class="c15"><p class="c8"><span class="c0">1</span></p></td><td class="c15"><p class="c8"><span class="c0">50&#1405;L</span></p></td><td class="c15"><p class="c8"><span class="c0">125&#1405;L</span></p></td><td class="c15"><p class="c8"><span class="c0">4C</span></p></td></tr><tr><td class="c15"><p class="c8"><span class="c0">2</span></p></td><td class="c15"><p class="c8"><span class="c0">50&#1405;L</span></p></td><td class="c15"><p class="c8"><span class="c0">125&#1405;L</span></p></td><td class="c15"><p class="c8"><span class="c0">15C</span></p></td></tr><tr><td class="c15"><p class="c8"><span class="c0">3</span></p></td><td class="c15"><p class="c8"><span class="c0">NONE</span></p></td><td class="c15"><p class="c8"><span class="c0">125&#1405;L</span></p></td><td class="c15"><p class="c8"><span class="c0">4C</span></p></td></tr><tr><td class="c15"><p class="c8"><span class="c0">4</span></p></td><td class="c15"><p class="c8"><span class="c0">NONE</span></p></td><td class="c15"><p class="c8"><span class="c0">125&#1405;L</span></p></td><td class="c15"><p class="c8"><span class="c0">15C</span></p></td></tr></tbody></table><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">12. Incubate overnight (16 hrs) at 37C.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/20/2010</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">Cold shock:</span></p><p class="c1"><span class="c0">1. Place places in appropriate temperature incubators (4C or 15C), and wait to see expression of RFP. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Results: One red colony observed on IPTG plate at 4C at 9:20 am.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/23/2010</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">Cold shock results:</span></p><p class="c1"><span class="c0">One red colony on IPTG plate at 4C (first observed at 9:20 am on 8/20/2010) and one red colony observed on IPTG at 15C (8/23/2010).</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/23/2010</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Purpose: try to maximize expression of mRFP. </span></p><p class="c1"><span class="c0 c2">Making a &ldquo;starter&rdquo; culture:</span></p><p class="c1"><span class="c0">1. From 4C NON-IPTG plate, take a white colony and place in approx. 3mL of LB/CARB media.</span></p><p class="c1"><span class="c0">2. Incubate with shaking overnight at 37C.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/24/2010</span></p><p class="c1"><span class="c0">Purpose: from starter culture monitor OD to obtain OD</span><span class="c11">600</span><span class="c0"> of 0.4-0.5.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. From starter culture take 50mL liquid culture and add 50uL CARB in a large Erlenmeyer flask. </span></p><p class="c1"><span class="c0">2. To this, add 1mL from starter culture and begin monitoring OD (for each aliquot take 1mL for measuring OD); maintain flask at 37C incubation with shaking. Keep in mind that e-coli doubling time is 20 minutes.</span></p><p class="c1"><span class="c0">3. Put on incubator/shaker at 11:20 am, with initial total volume of 51mL, and OD</span><span class="c11">600</span><span class="c0"> = 0.0.</span></p><table cellpadding="0" cellspacing="0" class="c19"><tbody><tr><td class="c15"><p class="c8"><span class="c0">aliquot</span></p></td><td class="c15"><p class="c8"><span class="c0">volume remaining in flask</span></p></td><td class="c15"><p class="c8"><span class="c0">time</span></p></td><td class="c15"><p class="c8"><span class="c0">OD</span><span class="c11">600</span></p></td></tr><tr class="c23"><td class="c15"><p class="c8"><span class="c0">1</span></p></td><td class="c15"><p class="c8"><span class="c0">50mL</span></p></td><td class="c15"><p class="c8"><span class="c0">11:45am</span></p></td><td class="c15"><p class="c8"><span class="c0">0.08</span></p></td></tr><tr class="c23"><td class="c15"><p class="c8"><span class="c0">2</span></p></td><td class="c15"><p class="c8"><span class="c0">49mL</span></p></td><td class="c15"><p class="c8"><span class="c0">12:45pm</span></p></td><td class="c15"><p class="c8"><span class="c0">0.32</span></p></td></tr><tr class="c23"><td class="c15"><p class="c8"><span class="c0">3</span></p></td><td class="c15"><p class="c8"><span class="c0">48mL</span></p></td><td class="c15"><p class="c8"><span class="c0">1:00pm</span></p></td><td class="c15"><p class="c8"><span class="c0">0.56</span></p></td></tr></tbody></table><p class="c8"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">4. Put incubator at 15C. During the cooling period, the flask was kept at 6C to ensure that the colony quit growing. Final OD was 0.57 before putting in the incubator (without shaking) for 30 minutes. (Time in: 1:30 pm). Total volume of the flask at this point was 47mL.</span></p><p class="c1"><span class="c0">5. At 2:00 pm, remove flask from incubator and IPTG (100mM) to a final concentration between 0.1 and 1mM (choose 0.25 mM). &nbsp;For a final concentration of 0.25mM IPTG in a 47mL solution, add 117.5uL of IPTG (100mM stock).</span></p><p class="c1"><span class="c0">6. &nbsp;Continue culture with shaking at 15C for 24 hours. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/25/2010</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0">Results from 8/24/2010: expression unsuccessful. </span></p><p class="c1"><span class="c0">Start with fresh colonies from the plates from 8/19/2010. &nbsp;Since we know the mRFP made it successfully into the pCold vector on the two colonies that turned red, try to make starter cultures from those. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Making a &ldquo;starter&rdquo; culture:</span></p><p class="c1"><span class="c0">1. From 4C and 15C IPTG plate, take a red colony and place in approx. 3mL of LB/CARB media. (3mL LB + 3uL CARB). </span></p><p class="c1"><span class="c0">2. Incubate the two tubes with shaking overnight at 37C. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Plan for 8/26/2010: Make cultures as we did on 8/24/2010 again, monitoring the concentration of cells. &nbsp;Then run a miniprep to ensure that we have the mRFP inserted into the pCold vector.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/26/2010</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0">Successful growth of starter colonies. Repeat process from 8/24/2010 until cell concentration is OD</span><span class="c11">600</span><span class="c0"> = 0.4 to 0.5.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. Prepare 2 flasks (1 for each starter colony) acccording to directions from 8/24/2010. </span></p><p class="c1"><span class="c0">2. Repeat procedure from 8/24/2010. (final concentration of cells in each flask was approx. OD600 = 0.4). Amount of (100mM) IPTG added: 125uL to each flask.</span></p><p class="c1"><span class="c0">3. Incubate with shaking overnight at 15C.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/27/2010</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0">WE GOT RED FLASKS!!!!!!!!!!!!</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Now, we&rsquo;re going to do a Miniprep and run an experiment analogous to what we did on 8/19/2010.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Miniprep </span><span class="c0">of 2 cultures: one inoculated from a fluorescent colony on the 4&ordm; C plate; one inoculated from a fluorescent colony on the 15&ordm; C. &nbsp;</span></p><ol class="c4"><li class="c14" value="1"><span class="c0">Protocol from 8/12/2010</span></li><li class="c14"><span class="c0">Concentrations: </span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><table cellpadding="0" cellspacing="0" class="c19"><tbody><tr><td class="c36"><p class="c8 c17"><span class="c0">pCOLD+mRFP (4&ordm; C)</span></p></td><td class="c36"><p class="c8 c17"><span class="c0">41.2 ng/uL</span></p></td></tr><tr><td class="c36"><p class="c8 c17"><span class="c0">pCOLD+mRFP (15&ordm; C)</span></p></td><td class="c36"><p class="c8 c17"><span class="c0">67.1 ng/uL</span></p></td></tr></tbody></table><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Transformation</span><span class="c0"> of BL21 by pCOLD+mRFP (from 4C &nbsp;and 15&ordm;C cultures)</span></p><ol class=""><li class="c3" value="1"><span class="c0">Plasmid, cells should be on ice initially</span></li></ol><ol class="c29"><li class="c3" value="1"><span class="c0">+ 1uL plasmid into 20 uL of competent cells (BL21)</span></li><li class="c3"><span class="c0">Sit on ice, 10 mins</span></li><li class="c3"><span class="c0">place in 42&ordm; water bath, 30 seconds </span></li><li class="c3"><span class="c0">place in ice, 2 minutes</span></li><li class="c3"><span class="c0">+250 uL LB broth</span></li><li class="c3"><span class="c0">Plate: spread 100 uL onto plate, use spatula to spread around until resistance is felt. </span></li></ol><ol class="c34"><li class="c14" value="1"><span class="c0">I plated an IPTG plate and a non-ITPG plate</span></li><li class="c14"><span class="c0">The IPTG plate had </span><span class="c0 c7">25 uL of 0.1 M IPTG</span><span class="c0"> </span></li></ol><ol class=""><li class="c3" value="7"><span class="c0">Plates were left (upside down) on bench at room temperature (since the starter culture is not needed until Sunday, and these cells were transformed today, Friday)</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">For Next Time: </span></p><p class="c1"><span class="c0">Sunday: Start 5 mL culture with one fluorescent colony of pCOLD+mRFP</span></p><p class="c1"><span class="c0">Monday: Protein Expression under various conditions (i.e. cold, iptg, cold+iptg) in 5x50mL flasks. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/29/2010</span></p><p class="c1"><span class="c0 c7">Results </span><span class="c0">from the plates of 8.27.2010 transformation (transformed BL21 with pCOLD+mRFP):</span></p><p class="c16"><span class="c0 c7">non-IPTG plat</span><span class="c0">e- growth, many colonies, some are slightly red</span></p><p class="c16"><span class="c0 c7">IPTG plate</span><span class="c0">- growth, many colonies, many are red, there are smaller red colones in the center along with larger normal ones; the last observation. Ryan suggested that it may indicate iptg stressed the cells in the middle (since IPTG could be more concentrated in middle).</span></p><p class="c16"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">What I did:</span></p><p class="c1"><span class="c0">MAKING A STARTER Culture: I picked a slightly red colony from the non-iptg plate and innoculated 5 ml of LB+Carboamp with it.</span></p><p class="c1"><span class="c0">Also, I put the iptg plate into the 15&ordm; C incubator and left it overnight </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/30/2010 &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Margo, Scott, Debika, Christian, and Christina</span></p><p class="c1"><span class="c0">Observations: the non-iptg had more red colonies than it previously did on 8/29/2010</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Induction experiment: inducing pCOLD+ mRFP under 4 different conditions (in BL21)</span></p><table cellpadding="0" cellspacing="0" class="c19"><tbody><tr><td class="c18"><p class="c8"><span class="c0">IPTG</span></p></td><td class="c31"><p class="c8"><span class="c0">-</span></p></td><td class="c42"><p class="c8"><span class="c0">-</span></p></td><td class="c30"><p class="c8"><span class="c0">+</span></p></td><td class="c28"><p class="c8"><span class="c0">+</span></p></td></tr><tr><td class="c18"><p class="c8"><span class="c0">15 degrees celsius</span></p></td><td class="c31"><p class="c8"><span class="c0">+</span></p></td><td class="c42"><p class="c8"><span class="c0">- </span></p></td><td class="c30"><p class="c8"><span class="c0">-</span></p></td><td class="c28"><p class="c8"><span class="c0">+</span></p></td></tr></tbody></table><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">We added 1ml of starter solution to 50 ml of LB media and 50 ul of Carb. Put it in the 37C incubator shaker for an hour and a half. Checked OD levels after 1.5hrs for one flask. Since it was at .3, we checked again in 10 minutes. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">After 10 minutes, the ODs values were as follows:</span></p><p class="c1"><span class="c0">&nbsp;</span></p><table cellpadding="0" cellspacing="0" class="c19"><tbody><tr><td class="c18"><p class="c8"><span class="c0">IPTG</span></p></td><td class="c39"><p class="c8"><span class="c0">-</span></p></td><td class="c40"><p class="c8"><span class="c0">-</span></p></td><td class="c20"><p class="c8"><span class="c0">+</span></p></td><td class="c41"><p class="c8"><span class="c0">+</span></p></td></tr><tr class="c23"><td class="c18"><p class="c8"><span class="c0">15 degrees celsius</span></p></td><td class="c39"><p class="c8"><span class="c0">+</span></p></td><td class="c40"><p class="c8"><span class="c0">-</span></p></td><td class="c20"><p class="c8"><span class="c0">-</span></p></td><td class="c41"><p class="c8"><span class="c0">+</span></p></td></tr><tr><td class="c18"><p class="c8"><span class="c0">OD value</span></p></td><td class="c39"><p class="c8"><span class="c0">.51</span></p></td><td class="c40"><p class="c8"><span class="c0">.47</span></p></td><td class="c20"><p class="c8"><span class="c0">.47</span></p></td><td class="c41"><p class="c8"><span class="c0">.52</span></p></td></tr></tbody></table><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">We wanted OD values to be @ ~0.4 (we got this number from the pCold manual). Added IPTG to one flask and put it in the 37C incubator w/ shaking along with another flask that had no IPTG.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Took the two other flasks and put it in the 15 degrees incubator shaker. After half an hour, we added IPTG to one of the flasks at 15 degrees, and left all 4 flasks in the respective incubators overnight. &nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">DMSO Stock</span><span class="c0"> (General protocol, applied here to pCOLD+mRFP containing cells from 8/30/2010)<br>Add to a cyrovial:</span></p><p class="c1"><span class="c0">900 uL starter culture (from 8/30/2010 which was already fluorescent)</span></p><p class="c1"><span class="c0">100 uL DMSO</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Vortex 5 seconds</span></li><li class="c3"><span class="c0">Put in -80c freezer (Gaucher lab)</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Transformation</span><span class="c0"> of BL21 by pCOLD+mRFP (from 4&ordm; C and 15&ordm;C cultures)</span></p><ol class="c10"><li class="c3" value="2"><span class="c0">Plasmid, cells should be on ice initially</span></li></ol><p class="c1"><span class="c0">1. 1 hour before transformation, spread </span><span class="c0 c7">25 uL of 0.1 M IPTG</span><span class="c0"> onto one plate</span></p><p class="c1"><span class="c0">2. + 1uL (1:1, ligation product) plasmid into 20 uL of competent cells (BL21) (edit:9.1.2010 the plasmid and cells should be on ice 15 mins before you start 2-8)</span></p><p class="c1"><span class="c0">3. Sit on ice, 10 mins</span></p><p class="c1"><span class="c0">4. place in 42&ordm;C water bath, 30 seconds </span></p><p class="c1"><span class="c0">5. place in ice, 2 minutes</span></p><p class="c1"><span class="c0">6. +250 uL LB broth (edit 9/1/2010: &nbsp;and wait 30 minutes @ 37C- tape down in incubator)</span></p><p class="c1"><span class="c0">7. Plate: spread 100 uL onto plate, use spatula to spread around until resistance is felt. </span></p><ol class=""><li class="c14" value="1"><span class="c0">I plated an IPTG plate and a non-ITPG plate</span></li><li class="c14"><span class="c0">The IPTG plate had </span><span class="c0 c7">25 uL of 0.1 M IPTG</span><span class="c0"> </span></li></ol><p class="c1"><span class="c0">8. Plates were left (upside down) in 37&ordm;C incubator overnight, to be examined on Tues 31 Aug.</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Note: Plate in incubator in room 218. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">8/31/2010</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c7">Results from the four flasks incubated overnight:</span></p><p class="c1"><span class="c0">+IPTG 37 C: Deep red cultures (color seen within an hour)</span></p><p class="c1"><span class="c0">no IPTG 37 C: No color</span></p><p class="c1"><span class="c0">+IPTG 15 C: Light red cultures (color seen the next morning)</span></p><p class="c1"><span class="c0">no IPTG 15 C: No color</span></p><p class="c1"><span class="c0">Contents of flasks bleached and discarded.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">pCold in solution doesn&rsquo;t require cold shock for expression, just IPTG. However, as seen from 8/29, pCold without IPTG colonies turned red when plated. Ryan noted that cells respond differently when in bulk vs. colonies, though these differences can&rsquo;t really be generalized. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Results from plates from the transformation conducted on 8/30/2010:</span></p><p class="c1"><span class="c0">No colonies were observed on the plates left in the incubator, cause: lack of incubation (essential for recovery after LB added) during transformation. During all transformations, the incubation step is crucial. Plates discarded.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Plan: </span></p><p class="c1"><span class="c0">1. Make more media.</span></p><p class="c1"><span class="c0">2. Make more plates.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Making LB media and plates</span></p><p class="c1"><span class="c0">To make 1L LB (Lysogeny broth) medium: </span></p><p class="c1"><span class="c0">5g Yeast abstract</span></p><p class="c1"><span class="c0">10g Tryptone</span></p><p class="c1"><span class="c0">5g NaCl</span></p><p class="c1"><span class="c0">Fill up to 1L with dH2O.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">To make solid plates, follow the above recipe and add 16g agar per 1L right before autoclaving.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Prepared 2L of LB media, 1L to be used as liquid broth and 1L to make plates.</span></p><p class="c1"><span class="c0">Let cool at room temp.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">For plate preparation:</span></p><p class="c1"><span class="c0">Add 1:1000 antibiotic (CARB - plates marked with blue line) after autoclaving and cooling.</span></p><p class="c16"><span class="c0">-1mL antibiotic per 1L media</span></p><p class="c1"><span class="c0">Pipette 25mL into each plate.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Plan for 9/1/10:</span></p><p class="c1"><span class="c0">1. Retransform from 15C colonies (not forgetting incubation!) from a miniprep.</span></p><p class="c1"><span class="c0">2. Grow up cell cultures of pCOLD+mRFP run the following 4 conditions (as with the bulk solutions observed this morning):</span></p><p class="c1"><span class="c0">&nbsp;</span></p><table cellpadding="0" cellspacing="0" class="c19"><tbody><tr class="c23"><td class="c26"><p class="c8"><span class="c0">IPTG</span></p></td><td class="c26"><p class="c8"><span class="c0">-</span></p></td><td class="c26"><p class="c8"><span class="c0">-</span></p></td><td class="c26"><p class="c8"><span class="c0">+</span></p></td><td class="c35"><p class="c8"><span class="c0">+</span></p></td></tr><tr class="c23"><td class="c26"><p class="c8"><span class="c0">15 degrees celsius</span></p></td><td class="c26"><p class="c8"><span class="c0">+</span></p></td><td class="c26"><p class="c8"><span class="c0">- (37 c)</span></p></td><td class="c26"><p class="c8"><span class="c0">- (37 c)</span></p></td><td class="c35"><p class="c1"><span class="c0">+</span></p></td></tr></tbody></table><p class="c8"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Further, another colony will be plated without IPTG and left at room temp. This might allow us to determine if there are repeatable differences between plate and bulk bacteria behavior. The red color from warm, NON-IPTG plates leads us to think that we might see red without IPTG, and the extra room-temp control is determine if the response is temp-dependent.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/1/2010</span></p><p class="c1"><span class="c0">11 am: Prepared IPTG plate for transformation (25 uL IPTG) (Scott)</span></p><p class="c1"><span class="c0">Margo, Scott, Mitesh, Gita, and Debika</span></p><p class="c1"><span class="c0">12 pm: Performed another transformation following protocol from 08/30/10. We transformed pcold+mrfp (pcold+mrfp plasmid, the 15c one from 8.27.2010). This time, we put both the non-iptg plate and iptg plate into a 15c incubator. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/2/2010</span></p><p class="c1"><span class="c0">Results: no cells grew on either plate from 9/1/2010.</span></p><p class="c1"><span class="c0">Thew plates away on 9/7/2010.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">3 pm: Gita and Christian</span></p><p class="c1"><span class="c0 c7">Objective:</span><span class="c0"> Determine if pCold is activated at intermediate concentrations of IPTG</span></p><p class="c1"><span class="c0">Performed serial dilution of IPTG to make 1 mM, 10uM, and 100nM, 1mL stocks. Grew bacteria from DMSO stock starter-culture from 8/30 in test tubes and incubated in 37C and 15C shakers.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><table cellpadding="0" cellspacing="0" class="c19"><tbody><tr><td class="c24"><p class="c8"><span class="c0">&nbsp;</span></p></td><td class="c24"><p class="c8"><span class="c0">0 IPTG &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;(1 &amp; 1&rsquo;)</span></p></td><td class="c24"><p class="c8"><span class="c0">9.99e-6 M IPTG (2 &amp; 2&rsquo;)</span></p></td><td class="c24"><p class="c8"><span class="c0">9.99e-8 M IPTG (3 &amp; 3&rsquo;)</span></p></td><td class="c24"><p class="c8"><span class="c0">9.99e-10 M IPTG (4 &amp; 4&rsquo;)</span></p></td></tr><tr><td class="c24"><p class="c8"><span class="c0">37 C (#&rsquo;)</span></p></td><td class="c24"><p class="c8"><span class="c0">&nbsp;</span></p></td><td class="c24"><p class="c8"><span class="c0">&nbsp;</span></p></td><td class="c24"><p class="c8"><span class="c0">&nbsp;</span></p></td><td class="c24"><p class="c8"><span class="c0">&nbsp;</span></p></td></tr><tr><td class="c24"><p class="c8"><span class="c0">15 C (#)</span></p></td><td class="c24"><p class="c8"><span class="c0">&nbsp;</span></p></td><td class="c24"><p class="c8"><span class="c0">&nbsp;</span></p></td><td class="c24"><p class="c8"><span class="c0">&nbsp;</span></p></td><td class="c24"><p class="c8"><span class="c0">&nbsp;</span></p><p class="c8"><span class="c0">&nbsp;</span></p></td></tr></tbody></table><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9.3.2010</span></p><p class="c1"><span class="c0">To do:</span></p><p class="c1"><span class="c0">-reconstitute aox, transform using NovaBlue</span></p><p class="c1"><span class="c0">-also, transform Ompa, hybB (check cells to transform into)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Experimental things:</span></p><p class="c1"><span class="c0">Plated some BL21 onto a plate that was made by iGEM, to test if something is wrong with the plates</span></p><p class="c1"><span class="c0">Plate labeled: 9.3.2010 Igem BL21</span></p><p class="c1"><span class="c0">Threw plates away on 9/7/2010.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Tuesday 9/7/2010</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c7">Where are the results from the experiment on 9/2/2010? </span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0">Results from 9/3/2010: </span></p><p class="c1"><span class="c0">No growth on the plate from 9/3/2010.</span><span class="c0 c7"> BL21 cells can not grow by themselves on plates with antibiotic. </span><span class="c0">The vectors we normally add contain antibiotic resistance genes to select for the colonies with our genes, BL21 by themselves don&rsquo;t contain these resistance genes. &nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Note that the &ldquo;Experimental things&rdquo; notes on 9/3/2010 is NOT experimental procedure. Please write completely and explicitly all steps.. even when just plating cells. </span><span class="c0">For example: How much of the BL21 cells were used? Were they frozen? Were they thawed? If so, how were they thawed? Water bath? Room temp? Pocket? Was anything else added? What temperature were they kept at? Where were plates kept and for how long? What was the plate labelled? What to do with them next? etc. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">We need experimental procedure and results at a MINIMUM. We should also have purpose, summaries of results, implications, hypotheses and even background information (example - why use BL21 instead of Novablue?). &nbsp;This should keep everybody up-to-date and help with coordination so people aren&rsquo;t backtracking to figure out what people did and why. </span></p><p class="c1"><span class="c0">-Christina and Rob</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">We have the iGem plates containing dry hybB, we have regular ompA, and we now have dry Aox genes. We want to grow up many copies of these, so we need to transform and grow cultures of them. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">We began by reconstituting (adding water) to all the dry DNA:</span></p><p class="c1"><span class="c0">50 uL for both AOX1a and AOX1b tubes</span></p><p class="c1"><span class="c0">10 uL to the hybB well (iGem Plate 2, well 7M) [observed an orange color solution]</span></p><p class="c1"><span class="c0">10 uL to the pSB1A3 backbone vector (iGem Plate 1, well 1C) [observed an orange color solution]</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">5 transformations were performed to grow future stocks: </span></p><p class="c1"><span class="c0">1. AOX1a in Novablue</span></p><p class="c1"><span class="c0">2. AOX1b in Novablue</span></p><p class="c1"><span class="c0">3. ompA in Novablue</span></p><p class="c1"><span class="c0">4. hybB in Novablue</span></p><p class="c1"><span class="c0">5. pSB1A3 in Novablue</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Heat shock transformation of the plasmids into our bacteria</span></p><p class="c1"><span class="c0">10 &#1405;L Nova Blue cells + 5 &#1405;L of each target plasmid</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. Left cells and ligation reaction products on ice.</span></p><p class="c1"><span class="c0">2. Added plasmid (5&#1405;L) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).</span></p><p class="c1"><span class="c0">3. Left cells on ice for 10-30 min </span></p><p class="c1"><span class="c0">4. Applied heat shock of 45 seconds in 42C bath.</span></p><p class="c1"><span class="c0">5. Put tubes on ice for 2 min. </span></p><p class="c1"><span class="c0">6. Added 250 &#1405;L of LB (room temp.)</span></p><p class="c1"><span class="c0">7. Incubated 1 hour at 37 C</span></p><p class="c1"><span class="c0">8. Plated 100&#1405;L, then remaining amount on separate LB/CARB plates.</span></p><p class="c1"><span class="c0">9. Incubated overnight at 37C. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">It was noted that several plates had heavy condensation- not sure if that might affect colony growth/development, so all were placed in the cell culture hood after plating to dry off the plates.</span></p><p class="c1"><span class="c0">The resulting plates (10 plates ttl, 2 for each transformation) were labeled and placed inside the 37C incubator in the iGem lab. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">For next time:</span></p><p class="c1"><span class="c0">Colonies should be selected for each of the 5 transformations, cell cultures for each should be grown in LB media, and incubated overnight. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/8/2010</span></p><p class="c1"><span class="c0 c7">Results</span></p><ol class="c10"><li class="c3" value="1"><span class="c0 c7">Plates from the transformations on 9/7/2010 all had growth</span><span class="c0">. The aoxa/b plates had many colonies (similar to a &ldquo;lawn&rdquo; of growth). Ryan suggested that for plasmids, we reduce the amount of plasmid we add to the cells during transformation (from 5uL to 1 uL). Transformations with plasmids require only 25-50 ng of DNA. </span></li><li class="c3"><span class="c0">Replate using triple smear technique (see below) to get distinct colonies.</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Goals:</span></p><p class="c1"><span class="c0">Pick 1 colony from each of the plates from the transformations done 9/7/2010, inoculate a 5 ml of LB+Carb in Falcon </span><span class="c0 c7">round bottom </span><span class="c0">tube. Grow these overnight @37c. Then on 9/9/2010, we can do 5 minipreps and 5 cryostocks (a.k.a DMSO stocks, where we freeze cells @-80c) on the starter cultures. The end result will be isolated plasmids of hyBb, AOXa/b, pSB1A3, and ompA. </span></p><p class="c1"><span class="c0">Actions:</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">I moved the plates from 9/7/2010 from 37C into the 4C refrigerator. Plates last longer in the refrigerator- at 37, they will overgrow!</span></li><li class="c3"><span class="c0">I created liquid cultures (per protocol listed below on 9/8/2010) 10am-11am for each of the transformants. They are at RT now and will place them in incubator around 5pm. </span></li></ol><p class="c1"><span class="c0">Liquid Cultures</span></p><ol class=""><li class="c3" value="3"><span class="c0">+5mL LB into 5 50mL eppendorfs. (changed to falcon round bottom tubes appropriate for incubator/shaker).</span></li></ol><ol class="c10"><li class="c3" value="1"><span class="c0">+50 uL 1000x Carb (antibiotic)</span></li><li class="c3"><span class="c0">Using a toothpick, gently scrape off and pick 1 discrete colony from a plate and put it into the falcon round bottom tube containing the LB+Carb.</span></li><li class="c3"><span class="c0">Put in incubator shaker 37C overnight (time on: 3:30 pm 9/8/10).</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">For Future/tomorrow:</span></p><p class="c1"><span class="c0">We are preparing liquid culture since we want to perform minipreps on the cells, isolate plasmids, and also create cryostocks of the transformed cells future use. Perform all of these tomorrow!</span></p><p class="c1"><span class="c0">Scott</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">2pm: Gita and Debika</span></p><p class="c1"><span class="c0">Aliquoting the primers: all need to &nbsp;b r PCR. </span></p><p class="c1"><span class="c0">1. centrifuge the tubes for 1 minute at 2000 rpm to get all of DNA at the bottom of the tube. </span></p><p class="c1"><span class="c0">2. add autoclaved water to dilute. ex. &nbsp;36.2 nM --&gt; add 362 microliters of water</span></p><p class="c1"><span class="c0">3. vortex, wait for 20 min, revortex</span></p><p class="c1"><span class="c0">4. store in freezer at -20 C</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">The plates grown up on 9/7/2010 do not contain discrete colonies, as the plates were wet when the cells went on. &nbsp;In order to ameliorate this problem, we will do a triple smear and replate so we can obtain distinct colonies. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Richard showed us the triple smearing technique. Christina placed all plates made yesterday under the hood for them to dry up. After an hour, she will check back on them and perform triple smearing. </span></p><p class="c1"><span class="c0">Triple Smearing Technique:</span></p><p class="c1"><img height="469.0" src="images/image2.png" width="609.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">All of the plates that we have currently in the 4C fridge need to be dried in the hood before use.</span><span class="c0">.. When we originally made them, they weren&rsquo;t allowed to cool long enough (should have been about 1 hour) before putting them in the fridge. &nbsp;As a result, the plates are pretty wet. </span><span class="c0 c7">Dry them first. (Note: 9/10/10 - all plates have been dried).</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Replating using smearing technique. Plates are labelled with: </span><span class="c0 c7">&lsquo;plasmid&rsquo;:novablue; 9/8/2010, iGEM</span><span class="c0">. </span></p><p class="c1"><span class="c0">Placed in the 37C incubator (in iGEM lab) overnight. </span></p><p class="c1"><span class="c0">Note: plates discarded on 9/9/2010.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/9/2010</span></p><p class="c1"><span class="c0 c7">Results from 9/8/2010:</span></p><p class="c1 c9"><span class="c0 c7">A) Triple smear: </span></p><p class="c1 c9"><span class="c0">Too many cells. Need to retry this technique. Plates thrown out.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1 c9"><span class="c0 c7">B) Starter colonies:</span></p><p class="c1 c9"><span class="c0">Successful growth of starter colonies prepared on 9/8/2010. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Procedure:</span></p><p class="c1 c9"><span class="c0 c7">A) Triple smear replating (hybB, ompA, Aox1a, Aox1b, pSB1A3):</span></p><p class="c1 c9"><span class="c0">1. Dry all plates in hood. </span></p><p class="c1 c9"><span class="c0">2. Try the triple smear replating technique again. Replate as on 9/8/2010 again using triple smear technique - too many cells were gathered on 9/8/2010, those plates were thrown out. &nbsp;Make sure to obtain a very small amount of cells. </span></p><p class="c1 c9"><span class="c0">&nbsp;</span></p><p class="c1 c9"><span class="c0 c7">B)</span><span class="c0"> </span><span class="c0 c7">Remake some starter colonies using distinct colonies from original plates (ompA, hybB, Aox1a, Aox1b) on 9/7/2010. </span></p><p class="c1 c9"><span class="c0">In an incubator tube add:</span></p><p class="c1 c9"><span class="c0">1. 5mL of LB media </span></p><p class="c1 c9"><span class="c0">2. add 5uL CARB</span></p><p class="c1 c9"><span class="c0">3. Add colony</span></p><p class="c1 c9"><span class="c0">4. incubate with shaking overnight at 37C</span></p><p class="c1 c9"><span class="c0">&nbsp;</span></p><p class="c1 c9"><span class="c0 c7">C) Miniprep of hybB</span><span class="c0"> (Gita, Christian, Scott)</span></p><p class="c1 c9"><span class="c0">Plasmid isolation protocol </span></p><ol class="c29"><li class="c3" value="1"><span class="c0">Pellet 1.5 mL of cells by centrifuging 2-3 mins @ 3k RPM, and pour off remaining liquid</span></li><li class="c3"><span class="c0">Resuspend pelleted bacterial cells in 250 &micro;L Buffer P1 and transfer to a microcentrifuge tube.</span></li><li class="c3"><span class="c0">Add 250 &micro;L Buffer P2 and mix thoroughly by inverting the tube 4 - 6 times.</span></li><li class="c3"><span class="c0">Add 350 &micro;L Buffer N3 and mix immediately and &nbsp;thoroughly by inverting the tube 4 &ndash; 6 times.</span></li><li class="c3"><span class="c0">Centrifuge for 10 mins at 13,000 rpm (~17,900 xg) in a table-top microcentrifuge.</span></li><li class="c3"><span class="c0">Apply the supernatants from step 5 to the spin column by pipetting.</span></li><li class="c3"><span class="c0">Centrifuge for 30 &ndash; 60 secs. Discard the flow-through.</span></li><li class="c3"><span class="c0">Wash spin column by adding 0.75 mL Buffer PE and centrifuging for 30 &ndash; 60 secs.</span></li><li class="c3"><span class="c0">Discard the flow-through, and centrifuge for an additional 2 min to remove residual wash buffer.</span></li><li class="c3"><span class="c0">Place the column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 30 &micro;L autoclaved water to the center of each spin column, let stand for 1 min, and &nbsp;centrifuge for 1 min. (NOTE: 50uL were added to elute)</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Results of miniprep </span></p><p class="c1"><span class="c0">HybB (two microcentrifuge tubes) is in the -20 in 218 (in a yellow container labeled Gaucher).</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Concentration</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;hybB 1: 159.1 ng/uL</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;hybB 2: 224.5 ng/uL</span></p><p class="c1"><span class="c0">Concentration obtained using nanospectrometer in Gaucher lab (226).</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Procedure:</span></p><p class="c1 c17"><span class="c0">1. Clean the sensor (center of the device where the arm meets the base) with a chemwipe.</span></p><p class="c1 c17"><span class="c0">2. Blank nanospec by pipetting 1uL of water (both water and pipet are next to the device) on to the sensor. Blow out the pipet so that the drop is hanging on the tip and touch it to the sensor rather than trying to drop the sample onto the sensor. Close the lever arm onto the sensor.</span></p><p class="c1 c17"><span class="c0">3. Click calibrate on the computer, wait for it to display 0ng/uL.</span></p><p class="c1 c17"><span class="c0">4. Clean sensor with Chemwipe.</span></p><p class="c1 c17"><span class="c0">5. Pipet 1uL of sample onto sensor, close lever arm.</span></p><p class="c1 c17"><span class="c0">6. Click measure and wait for sample reading.</span></p><p class="c1 c17"><span class="c0">7. Repeat 4-6 for each sample, clean sensor when done.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">For next time-</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Minipreps and cryostocks of the ompa, aoxa/b, pSB1A3 </span></li><li class="c3"><span class="c0">Start PCR steps for hyBb (getting it ready for the 5 constructs(</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/10/2010 </span><span class="c0">(Scott, Debika, Christina, Margo, Gita)</span></p><p class="c1"><span class="c0 c7">Results and Observations:</span></p><p class="c1"><span class="c0 c7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;A) Triple smear: </span><span class="c0">triple smear worked fine on the new plates with distinct colonies. Plates are in 4C fridge.</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c7">B) Starter colonies from 9/9/10:</span><span class="c0"> Don&rsquo;t appear very dense - interestingly the pSB1A3 starter colony didn&rsquo;t turn red like it did last time.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Experimental Procedure:</span></p><p class="c1"><span class="c0 c7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;A) Miniprep of Aox1a, Aox1b, ompA, pSB1A3 from starter colonies from 9/9/10.</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">Miniprep</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. Remove inoculation tubes from inoculation (37C shaker).</span></p><p class="c1"><span class="c0">2. Obtain P1 buffer from 4C refrigerator.</span></p><p class="c1"><span class="c0">3. Take centrifuge tubes and add 1.5mL on inoculated cells. </span></p><p class="c1"><span class="c0">4. Centrifuge at 3000 rpm (low) for 1-2 min. </span></p><p class="c1"><span class="c0">5. Spin until white pellet of cells forms at the bottom and liquid is more clear. </span></p><p class="c1"><span class="c0">6. Take off supernatant and discard. </span></p><p class="c1"><span class="c0">7. Repeat steps 4-6. </span></p><p class="c1"><span class="c0">8. Resuspend pelleted bacterial cells in 250&#1405;L P1 buffer. </span></p><p class="c1"><span class="c0">9. &nbsp;Add 250&#1405;L P2 buffer and invert 4-6 times (DO NOT VORTEX - doing so will shear DNA!)</span></p><p class="c1"><span class="c0">10. Add 350&#1405;L buffer N3 and immediately invert 4-6 times. </span></p><p class="c1"><span class="c0">11. Centrifuge for 10 min. at 13,000 rpm. </span></p><p class="c1"><span class="c0">12. Take supernatant and add to spin columns. </span></p><p class="c1"><span class="c0">13. Spin 30-60 sec. and discard flow through. </span></p><p class="c1"><span class="c0">14. Wash column with 750&#1405;L buffer PE and centrifuge 1 min. </span></p><p class="c1"><span class="c0">15. Discard flow through and centrifuge and additional minute.</span></p><p class="c1"><span class="c0">16. Please column into a clean 1.5mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">17. Elute DNA by adding 30&#1405;L dH2O. </span></p><p class="c1"><span class="c0">18. Let stand for 1 min., then centrifuge for 1 min. </span></p><p class="c1"><span class="c0">Note for miniprep- I did 3 tubes for each plasmid, and I used 50 uL of water to elute DNA from one tube, then I used that 50 uL of already eluted DNA to elute the other two in series. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Making gel for PCR</span></p><p class="c1"><span class="c0">1. Add 0.35g agarose to 35mL autoclaved water.</span></p><p class="c1"><span class="c0">2. Add 3.5mL 1X TBE</span></p><p class="c1"><span class="c0">3. &nbsp;Heat in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don&rsquo;t vaporize it, especially near your face!</span></p><p class="c1"><span class="c0">4. Add 38.5&#1405;L EtBr (edited from 45 uL, make 1000X)</span></p><p class="c1"><span class="c0">5. Pour gel and allow to harden. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">First Step for PCR</span><span class="c0"> (9/10/2010)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Labels on Tube:</span></p><p class="c1"><span class="c0"> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;1= HybB F,R</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2= AOX1a F,R </span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3= AOX1a F,R2</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4= AOX1b F,R</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5= AOX1b F,R2</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;6= Ompa F, R</span></p><p class="c1"><span class="c0 c2">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR Basic Recipe</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;template DNA</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 or more primers</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;dNTPs</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;buffer compatible with DNA polymerase being used</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DNA polymerase</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Today, we didn&rsquo;t have any RFP plasmid left, so we just did the following combinations:</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1 = HybB F, R primers with HybB template DNA</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 = AOX1a F, R primers with AOX1a template DNA</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3 = AOX1a F, R2 primers with AOX1a template DNA</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4 = AOX1b F, R primers with AOX1b template DNA</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5 = AOX1b F, R2 primers with AOX1b template DNA</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;6 = OmpA F, R primers with OmpA template DNA</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">The polymerase used in ALL tubes is the Phusion and the buffer used is the Phusion &nbsp;MF Reaction Buffer . All components listed are stored in the iGEM freezer.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">The steps for running PCR are as follows:</span></p><p class="c1"><span class="c0">1. Obtain PCR tubes (very small, string of 8 tubes) with lids (also strung 8 together). Label. Obtain components. Buffer and template can be thawed at room temperature; enzymes and primers should be thawed on ice just long enough to obtain liquid &amp; refrozen.</span></p><p class="c1"><span class="c0">2. Primers used are 1:10 dilution. We prepped them in Eppe tubes, 20 uL into 180 uL for a total of 200 uL, stored in the freezer. For use in reactions, to avoid contaminating stock by repeatedly drawing from it.</span></p><p class="c1"><span class="c0">3. Template concentration must be measured, and fall between 50 and 200 ng/uL. If prepared template does not fall in this range, adjust amount used in step 4e. below, and adjust water used in step 4a.</span></p><p class="c1"><span class="c0">4. Add liquid components, keeping primers, dNTPs, template, and polymerase on ice (NOTE: components in steps c, d, e can be added in any order. Just be sure a &amp; b go in first, and f goes in last.):</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;a. 14 uL H2O</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;b. 5 uL Phusion MF Reaction buffer</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;c. 2.5 uL forward primer &amp;</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp; &nbsp;2.5 uL reverse primer</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;d. 0.5 uL dNTP - (thawed &amp; kept on ice)</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;e. 0.25 uL template DNA</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;f. 0.25 uL polymerase enzyme, Phusion.</span></p><p class="c1"><span class="c0">5. Volume should total 25 uL. Seal lids. Store all leftover components at -20 degree freezer. Run in PCR cycler.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Instructions for running PCR thermocycler:</span></p><p class="c1"><span class="c0">1. The on/off switch is on the back right side. Turn it on. The machine has a graphic showing you how to open it. Basically, turn the top wheel counter-clockwise to open, push in.</span></p><p class="c1"><span class="c0">2. Our cycle program is saved under RUN -&gt; EAG1 -&gt; MEGAN -&gt; IGEM. Select. Tell it if you are using side A or B. Hit run.</span></p><p class="c1"><span class="c0">3. When prepping, it will ask for approximate sample size. Saying 25 or 30 uL is fine. Leave SampleID and User blank. Select Heat lids to 105 C.</span></p><p class="c1"><span class="c0">4. Load tubes, avoiding corner slots (if possible). Close lid, tighten clockwise until you feel minor resistance-- don&rsquo;t crank too hard.</span></p><p class="c1"><span class="c0">3. PCR cycler will hold at 12 degrees C after completion of cycles, until you pick it up (or someone else turns the machine off).</span></p><p class="c1"><span class="c0">4. When you are done, if no one else is using the machine, turn it off.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Results (9.10.2010)</span></p><p class="c1"><span class="c0">Miniprep-</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Concentrations (ng/uL)</span></p><p class="c1"><span class="c0">AOXa-350</span></p><p class="c1"><span class="c0">AOXb-313</span></p><p class="c1"><span class="c0">PSB1A3-107</span></p><p class="c1"><span class="c0">ompA- 80</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/11/2010</span></p><p class="c1"><span class="c0">Goals: Purify the PCR reactions and look at them on a gel</span></p><p class="c1"><span class="c0 c2">PCR purifcation of the PCR reactions from 9/10/2010</span></p><p class="c1"><span class="c0">1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)</span></p><p class="c1"><span class="c0">2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).</span></p><p class="c1"><span class="c0">3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">4. Discarded flow-through. Placed the column back in the same tube.</span></p><p class="c1"><span class="c0">5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.</span></p><p class="c1"><span class="c0">7. Placed the column in a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">8. To elute DNA, 30 &nbsp;&micro;L autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">We purified both strips 1 and 2 from the PCR last night, they are labeled this way in the freezer:</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1 HybB F, R</span></p><p class="c1"><span class="c0">1 AOX1a F, R</span></p><p class="c1"><span class="c0">1 AOX1a F, R2 </span></p><p class="c1"><span class="c0">1 AOX1b F, R</span></p><p class="c1"><span class="c0">1 AOX1b F, R2</span></p><p class="c1"><span class="c0">1 OmpA F, R</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">2 HybB F, R</span></p><p class="c1"><span class="c0">2 AOX1a F, R</span></p><p class="c1"><span class="c0">2 AOX1a F, R2 </span></p><p class="c1"><span class="c0">2 AOX1b F, R</span></p><p class="c1"><span class="c0">2 AOX1b F, R2</span></p><p class="c1"><span class="c0">2 OmpA F, R</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">As we were not certain that the PCR strip 1 had been successfully put together (may have doubled up on the buffer and neglected to add the enzyme), we decided to run the gel using just the samples from the PCR strip 2. We used only 5 uL of each, leaving the leftovers in the freezer for later reference or use.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Running an Agarose Gel to see reults of strip 2 PCR from 9/10/2010</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">After running PCR purification using QIAGEN kit (eluting at the end into water), prepared a gel with 12 wells. Prepared gel electrophoresis kit by placing fresh gel into well, and filling with 1X TBE up to the top of the gel.</span></li><li class="c3"><span class="c0">Laid out a strip of clean parafilm, made discrete dots of 1 uL of dye for each sample (excluding ladder) being prepared.</span></li><li class="c3"><span class="c0">Mixed 5 uL of DNA in water with 1 uL of loading dye for each sample, directly onto parafilm. Pulled up a total of 6 uL. Transferred to the gel in this order:</span></li></ol><ol class="c4"><li class="c14" value="1"><span class="c0">Lane 1: DNA Ladder (6 uL) (Pretty sure you mean 5 uL and not 56 uL. Edited by Debika @ 1:54 am on 9/12/10. Yes, thanks. Margo).</span></li><li class="c14"><span class="c0">Lane 2: 2 HybB F, R</span></li><li class="c14"><span class="c0">Lane 3: 2 AOX1a F, R</span></li><li class="c14"><span class="c0">Lane 4: 2 AOX1a F, R2 &nbsp;</span></li><li class="c14"><span class="c0">Lane 5: 2 AOX1b F, R</span></li><li class="c14"><span class="c0">Lane 6: 2 AOX1b F, R2</span></li><li class="c14"><span class="c0">Lane 7: 2 OmpA F, R</span></li><li class="c14"><span class="c0">Lane 8: DNA Ladder (5 uL)</span></li></ol><ol class=""><li class="c3" value="4"><span class="c0">Plugged in gel electrophoresis kit, ran at 100 V for ~ 25 min.</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Results 9/11/2010</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">We could not see bands for the PCR reactions, except for the 4th and 6th lanes (very faint bands, and not in the correct &ldquo;spot&rdquo;. </span></li></ol><p class="c1"><span class="c0 c7">For next time-</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">We need to troubleshoot why we couldn&rsquo;t see bands for the PCR reactions. </span></li></ol><ol class="c4"><li class="c14" value="1"><span class="c0">Possibilities:</span></li><li class="c14"><span class="c0">Use smaller wells (we used 20 uL wells)</span></li><li class="c14"><span class="c0">Use more DNA (we loaded 5 uL of PCR DNA)</span></li><li class="c14"><span class="c0">Redo the PCR reactions</span></li></ol><ol class="c10"><li class="c3" value="2"><span class="c0">Scan the Gel Picture </span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2 c7">9/13/2010</span></p><p class="c1"><span class="c0 c7">Christina, Christian, Scott, Debika, Margo</span></p><p class="c1"><span class="c0 c2">&nbsp;</span></p><p class="c1"><span class="c0">Started from new aliquots of primers, 1:10 dilution of primer stock in MilliQ H2O. Changes are </span><span class="c0 c7">bolded</span><span class="c0">.</span></p><p class="c1"><span class="c0">Reactions: HybB F+R, OmpA F+R, AOX1A F&amp;R, and AOX1B F&amp;R.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2 c7">New PCR protocol:</span></p><p class="c1"><span class="c0">26.5 uL H2O</span></p><p class="c1"><span class="c0">10 uL </span><span class="c0 c7">TaQ 5X Reaction buffer</span></p><p class="c1"><span class="c0">5 uL forward primer </span></p><p class="c1"><span class="c0">5 uL reverse primer</span></p><p class="c1"><span class="c0">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c1"><span class="c0 c7">2 uL</span><span class="c0"> template DNA</span></p><p class="c1"><span class="c0">0.5 uL polymerase enzyme, </span><span class="c0 c7">TaQ</span></p><p class="c1"><span class="c0 c7">Total Volume= 50 uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">NOTE: We multiplied the entire protocol by 2 to get </span><span class="c0 c7">50 &nbsp;uL total volume</span><span class="c0"> for this attempt</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c5">We also decided to load just the plasmids onto gel TODAY, to check that our template stocks are fine and were not the issue with the PCR failure this past weekend.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">We are reducing number of experiments at once (e.g. not all setups at once, just a few) - HybB, OmpA, AOX1A F&amp;R, and AOX1B F&amp;R.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">We are preparing two strips for PCR using this recipe and setup. We are then running them side-by-side in the PCR machine, one strip using the old cycle program, and one strip with two key modifications (suggested by Megan). We increased number of PCR cycles from 29 to 34 cycles, and reduced annealing temperature to 52 degrees. (If we go to low with the annealing temperature, we will be able to tell because we will see lots of bands on PCR.)</span></p><p class="c1"><span class="c0"></span><span class="c0 c2">Making gel for PCR</span></p><p class="c1"><span class="c0">1. Add 0.35g agarose to 35mL autoclaved water.</span></p><p class="c1"><span class="c0">2. Add 3.5mL 1X TBE</span></p><p class="c1"><span class="c0">3. &nbsp;Heat in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don&rsquo;t vaporize it, especially near your face!</span></p><p class="c1"><span class="c0">4. Add 38.5&#1405;L EtBr (edited from 45 uL, make 1000X)</span></p><p class="c1"><span class="c0">5. Pour gel and allow to harden. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Goals: Purify the PCR reactions and look at them on a gel</span></p><p class="c1"><span class="c0 c2">PCR purifcation of the PCR reactions from 9/10/2010</span></p><p class="c1"><span class="c0">1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)</span></p><p class="c1"><span class="c0">2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).</span></p><p class="c1"><span class="c0">3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">4. Discarded flow-through. Placed the column back in the same tube.</span></p><p class="c1"><span class="c0">5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.</span></p><p class="c1"><span class="c0">7. Placed the column in a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">8. To elute DNA, 30 &nbsp;&micro;L autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">note: unless we want to keep the DNA for future use (unless we NEED pure DNA), the purification step is not necessary. &nbsp;We could have just run the DNA without the purification step... EtBr is an intercalator that will only bind the DNA anyway.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR Results via Gel Electrophoresis</span></p><p class="c1"><span class="c0">Ran at 100 V for ~ 4 minutes, until dye was visible ~ 3/4 of the way across the gel. (This seems like it was too long!)</span></p><p class="c1"><span class="c0"> </span></p><p class="c1"><span class="c0">DNA was visible in all four lanes; two replicates of each lane (labeled A and H), with the A samples having been run with the &ldquo;old&rdquo; PCR cycle program, and the H samples having been run with the &ldquo;new&rdquo; PCR cycle program (as described by today&rsquo;s changes).</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. HybB with Forward and Reverse primers</span></p><p class="c1"><span class="c0">2. AOX1a with Forward and Reverse primers</span></p><p class="c1"><span class="c0">3. AOX1b with Forward and Reverse primers</span></p><p class="c1"><span class="c0">4. OmpA with Forward and Reverse primers</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">The A lanes and its DNA ladder were &nbsp;more clearly visible than the H lanes. The visible results are:</span></p><p class="c1"><span class="c0">1. Faint bands in A &amp; H at ~ 6,000 bp</span></p><p class="c1"><span class="c0">2. In A&amp; H, very strong bands at 700 bp, strong bands at 3,000 bp, somewhat weak but &nbsp;still obvious band at 2,000 bp.</span></p><p class="c1"><span class="c0">3. In A&amp; H, very strong bands at 700 bp, strong bands at 3,000 bp, somewhat weak but &nbsp;still obvious band at 2,000 bp.</span></p><p class="c1"><span class="c0">4. DNA may have &ldquo;run off&rdquo; the gel! In the H lane, distinct but faint band visible past the visible bands of the DNA ladder-- not sure how this should be interpreted.</span></p><p class="c1"><span class="c0 c2 c7">&nbsp;</span></p><p class="c1"><span class="c0 c7">(Pics from 9/11 and 9/13 were taped to the lab bench for group reference -- maybe get Megan and/or Richard to help us interpret.)</span></p><p class="c1"><span class="c0 c2 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2 c7">Starter cultures for cryostocks</span></p><p class="c1"><span class="c0 c2 c7">&nbsp;</span></p><p class="c1"><span class="c0">Made starter cultures (3uL CARB + 3mL LB + cells) from triple smear plates (9/10/2010), and put in the incubator for 24 hours.. &nbsp;Tomorrow (on 9/14/2010), make cryostocks from these starter cultures. Labelled according to insert.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2 c7">9/14/2010</span></p><p class="c1"><span class="c0">Results from 9/13/2010: &nbsp;The previous gel had fairly good AOX bands but after consulting with Richard we decided that the other samples weren&rsquo;t represented in the gel. Further, we should have seen primer bands near the end, so in future gels it&rsquo;s important not to let samples run off. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2 c7">Miniprep</span></p><p class="c1"><span class="c0">In order to prepare for another gel, a miniprep of the following samples from the starter cultures made on 9/13/2010 were run:</span></p><p class="c1"><span class="c0">1. PSB1A3</span></p><p class="c1"><span class="c0">2. AOX1a</span></p><p class="c1"><span class="c0">3. ompA</span></p><p class="c1"><span class="c0">4. hybB</span></p><p class="c1"><span class="c0">5. AOX1b</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">The contents were labeled and stored in the -20 freezer for further use in the gel. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2 c7">Crystocks</span></p><p class="c1"><span class="c0">Cryostocks were made from starter cultures grown on 9/13/2010. &nbsp;Two distinct colonies were taken from each plate - so there are duplicates of each. &nbsp;Stored in the -80C labelled: </span></p><p class="c1"><span class="c0">9-14 clg HybB (2)</span></p><p class="c1"><span class="c0">9-14 clg ompA (2)</span></p><p class="c1"><span class="c0">9-14 clg AOX1a (2)</span></p><p class="c1"><span class="c0">9-14 clg &nbsp;AOX1b (2)</span></p><p class="c1"><span class="c0">9-14 clg psb1A3 (2) [note: 1 of these starter cultures turned red, the other did not.]</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9.15.2010</span></p><p class="c1"><span class="c0 c2">Goals:</span><span class="c0"> Perform PCR on the new minipreps from 9/14/2010 (using new 1:10 aliquots of primers from 9/13/2010)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR Protocol</span></p><p class="c1"><span class="c0">26.5 uL H2O</span></p><p class="c1"><span class="c0">10 uL </span><span class="c0 c7">TaQ 5X Reaction buffer</span></p><p class="c1"><span class="c0">5 uL forward primer </span></p><p class="c1"><span class="c0">5 uL reverse primer</span></p><p class="c1"><span class="c0">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c1"><span class="c0 c7">2 uL</span><span class="c0"> template DNA</span></p><p class="c1"><span class="c0">0.5 uL polymerase enzyme, </span><span class="c0 c7">TaQ</span></p><p class="c1"><span class="c0 c7">Total Volume= 50 uL</span></p><p class="c1"><span class="c0">Notes: Wear gloves while doing the reaction. Keep all reagents on ice, including the PCR reactions. Add in the order of the protocol- get out the enzyme and place on ice right before you are about to use.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Reactions Done in PCR:</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">HyBb F,R</span></li><li class="c3"><span class="c0">OmpA F,R</span></li><li class="c3"><span class="c0">Aox1a F,R</span></li><li class="c3"><span class="c0">Aox1a F,R2</span></li><li class="c3"><span class="c0">Aox1b F,R</span></li><li class="c3"><span class="c0">Aox1b F,R2</span></li></ol><p class="c1"><span class="c0">Notes: We are using a master mix of Water, TAQ Buffer, DNTPS, and TAQ. Add this to all the tubes (everything on ice), then add all the DNA reagents.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2 c7">Results from nanospec:</span></p><p class="c1"><span class="c0 c2 c7">Sample&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Concentration (ng/uL)</span></p><p class="c1"><span class="c0">hybB&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;155.2 </span></p><p class="c1"><span class="c0">ompA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;70.2</span></p><p class="c1"><span class="c0">Aox1a&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;241.3</span></p><p class="c1"><span class="c0">Aox1b&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;253.2</span></p><p class="c1"><span class="c0">psb1A3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;53.6</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Making gel for PCR</span></p><p class="c1"><span class="c0">1. Add 0.35g agarose to 36 mL 1 x TBEautoclaved water.</span></p><p class="c1"><span class="c0">2. &nbsp;Heat in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don&rsquo;t vaporize it, especially near your face!</span></p><p class="c1"><span class="c0">3. Add 35 &#1405;L EtBr (edited from 45 uL, make 1000X)</span></p><p class="c1"><span class="c0">4. Pour gel and allow to harden. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/16/2010</span></p><p class="c1"><span class="c0 c2">Goals: </span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Interpret gel results of the PCR from 9/15/2010</span></li><li class="c3"><span class="c0">Perform PCR (from 9/15/2010) again, this time with:</span></li></ol><ol class="c4"><li class="c14" value="1"><span class="c0">PHUSION Polymerase/Buffer</span></li><li class="c14"><span class="c0">RFP</span></li></ol><p class="c1"><span class="c0 c2">What we did:</span></p><p class="c1"><span class="c0">(Christina, Rob)</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Performed PCR with Phusion </span></li><li class="c3"><span class="c0">Reactions: </span></li></ol><ol class="c34"><li class="c14" value="1"><span class="c0">HyBb F,R</span></li><li class="c14"><span class="c0">OmpA F,R</span></li><li class="c14"><span class="c0">Aox1a F,R</span></li><li class="c14"><span class="c0">Aox1a F,R2</span></li><li class="c14"><span class="c0">Aox1b F,R</span></li><li class="c14"><span class="c0">Aox1b F,R2</span></li><li class="c14"><span class="c0">RFP F,R</span></li><li class="c14"><span class="c0">RFP F2,R</span></li></ol><ol class=""><li class="c3" value="7"><span class="c0">Performed miniprep of pSB1A3 from cell culture grown on 9/15/2010 (non-red)</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR Protocol</span></p><p class="c1"><span class="c0">26.5 uL H2O</span></p><p class="c1"><span class="c0">10 uL </span><span class="c0 c7">PHUSION 5X Reaction buffer</span></p><p class="c1"><span class="c0">5 uL forward primer </span></p><p class="c1"><span class="c0">5 uL reverse primer</span></p><p class="c1"><span class="c0">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c1"><span class="c0 c7">2 uL</span><span class="c0"> template DNA</span></p><p class="c1"><span class="c0">0.5 uL polymerase enzyme, </span><span class="c0 c7">PHUSION</span></p><p class="c1"><span class="c0 c7">Total Volume= 50 uL</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">Miniprep of pSB1A3</span></p><p class="c1"><span class="c0">1. Remove inoculation tubes from inoculation (37C shaker).</span></p><p class="c1"><span class="c0">2. Obtain P1 buffer from 4C refrigerator.</span></p><p class="c1"><span class="c0">3. Take centrifuge tubes and add 1.5mL of inoculated cells. </span></p><p class="c1"><span class="c0">4. Centrifuge at 3000 rpm (low) for 1-2 min. </span></p><p class="c1"><span class="c0">5. Spin until white pellet of cells forms at the bottom and liquid is more clear. </span></p><p class="c1"><span class="c0">6. Take off supernatant and discard. </span></p><p class="c1"><span class="c0">7. Repeat steps 4-6. </span></p><p class="c1"><span class="c0">8. Resuspend pelleted bacterial cells in 250&#1405;L P1 buffer. </span></p><p class="c1"><span class="c0">9. &nbsp;Add 250&#1405;L P2 buffer and invert 4-6 times (DO NOT VORTEX - doing so will shear DNA!)</span></p><p class="c1"><span class="c0">10. Add 350&#1405;L buffer N3 and immediately invert 4-6 times. </span></p><p class="c1"><span class="c0">11. Centrifuge for 10 min. at 13,000 rpm. </span></p><p class="c1"><span class="c0">12. Take supernatant and add to spin columns. </span></p><p class="c1"><span class="c0">13. Spin 30-60 sec. and discard flow through. </span></p><p class="c1"><span class="c0">14. Wash column with 750&#1405;L buffer PE and centrifuge 1 min. </span></p><p class="c1"><span class="c0">15. Discard flow through and centrifuge and additional minute.</span></p><p class="c1"><span class="c0">16. Please column into a clean 1.5mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">17. Elute DNA by adding 30&#1405;L dH2O. </span></p><p class="c1"><span class="c0">18. Let stand for 1 min., then centrifuge for 1 min. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Cryostock of hybB cells and pSB1A3 </span><span class="c0">(from cultures on 9/15/2010)</span></p><p class="c1"><span class="c0">900 uL of cells</span></p><p class="c1"><span class="c0">100 uL DMSO</span></p><p class="c1"><span class="c0 c7">Total 1 mL</span></p><p class="c1"><span class="c0">Mix, place in -80c Freezer</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">For next time</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Confirm all the predicted sizes match up with the observed sizes on the gel of the PCR from 9/15/2010 </span></li><li class="c3"><span class="c0">If all are confirmed (Christina has confirmed, Christian and Scott confirmed hybb, ompa), then proceed with PCR purifications of 40 uL of each reaction, then RE digests, then ligations to construct each construct</span></li></ol><p class="c1"><span class="c0 c2">&nbsp;</span></p><p class="c1"><span class="c0 c7">SEPTEMBER 17, 2010</span></p><p class="c1"><span class="c0 c2">Goals:</span></p><p class="c1"><span class="c0">Since the PCR was run on 9/16 using Phusion (high fidelity), we need to:<br>A) Run gel (2 uL each)</span></p><p class="c1"><span class="c0">B) PCR purify (leftovers from the 50 uL stock= 48 uL)</span></p><p class="c1"><span class="c0">C) Nanospec</span></p><p class="c1"><span class="c0">D) Digest with restriction enzymes (runs overnight)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Protocols</span></p><p class="c1"><span class="c0 c2">&nbsp;</span></p><p class="c1"><span class="c0">Make a gel for running PCR products from 9/16/2010 to make sure the PCR was successful. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Making gel for PCR (1% agarose gels):</span></p><p class="c1"><span class="c0">Added 180 mL 1x TBE and 1.8g agarose</span></p><p class="c1"><span class="c0">Heated up until agarose was no longer visible</span></p><p class="c1"><span class="c0">Let cool (approx. 10 min) until there are NO MORE VAPORS</span></p><p class="c1"><span class="c0">Added 180 &#1405;L Ethidium bromide (1000X)</span></p><p class="c1"><span class="c0">Pour into gels and allow to harden (large wells hold about 35 mL).</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Note: it is ok to make 6 gels at once according to the recipe above and store them. It is not necessary to &ldquo;immediately&rdquo; use gels as long as they are stored properly. From now on, when you make 1% gels, just fill all 6 wells at once.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Gel of the PCR from 9/16/2010 was run today and imaged:</span></p><p class="c1"><span class="c0">Lane order: 100 bp ladder|aox1aFR|aox1aFR2|aox1bFR|apx1bFR2|HybB FR|ompa FR|RFP FR| RFP F2R|100 bp ladder</span></p><p class="c1"><span class="c0">volumes:</span></p><p class="c1"><span class="c0">loaded 4 ul of the aox reactions, 7 ul of ompa and hybB, and 5 ul of the RFP reactions</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR Purificatio</span><span class="c0">n </span></p><p class="c1"><span class="c0">1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)</span></p><p class="c1"><span class="c0">2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).</span></p><p class="c1"><span class="c0">3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">4. Discarded flow-through. Placed the column back in the same tube.</span></p><p class="c1"><span class="c0">5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.</span></p><p class="c1"><span class="c0">7. Placed the column in a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">8. To elute DNA, 30 &nbsp;&micro;L autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Nanospec results </span><span class="c0">(PCR from 9/16/2010)</span></p><p class="c1"><span class="c0">AOX1a-F,R = 99.3 ng/uL</span></p><p class="c1"><span class="c0">AOX1a-F,R2 = 50.0 ng/uL</span></p><p class="c1"><span class="c0">AOX1b-F,R = 48.5 ng/uL</span></p><p class="c1"><span class="c0">AOX1b-F,R2 = 42.1 ng/uL</span></p><p class="c1"><span class="c0">OmpA-F,R = 27.3 ng/uL</span></p><p class="c1"><span class="c0">RFP-F2,R = 10.8 ng/uL</span></p><p class="c1"><span class="c0">Hyb-F,R = 26.2 ng/uL</span></p><p class="c1"><span class="c0">RFP-F,R = 8.8 ng/uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Restriction Enzyme Digest General Process</span></p><p class="c1"><span class="c0">- check buffers (NEB site)</span></p><p class="c1"><span class="c0">-check if BSA required (NEB site)</span></p><p class="c1"><span class="c0">- Start w/ HybB and RFP (start @ step A) / pBS1A3 (start @ step D)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Recipe for : 50 uL RXN</span></p><p class="c1"><span class="c0">DNA (1-2 ug- based on nanospec results)</span></p><p class="c1"><span class="c0">1 uL each enzyme</span></p><p class="c1"><span class="c0">5 uL 10X buffer</span></p><p class="c1"><span class="c0">1 uL BSA(if needed)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Today we are digesting just the HybB-F,R and RFP-F,R in order to do the ligation on this simple construct. The rest of the PCR-ed, purified, and nanoscpec&rsquo;ed building blocks went into the freezer for later use.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">RE Digest Recipe for Hyb</span><span class="c0">B</span></p><p class="c1"><span class="c0">40 uL HybB-F,R (based on the nanospec results above)</span></p><p class="c1"><span class="c0">1 uL EcoRI</span></p><p class="c1"><span class="c0">1 uL NotI</span></p><p class="c1"><span class="c0">4.7 uL 10X buffer for EcoRI [Edit 9/17/2010. Only EcoRIbuffer)</span></p><p class="c1"><span class="c0">1 uL 10x BSA (based on this specfic RE mix-- BSA is required)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Place into an eppe tube, pipet gently to mix. Incubate at 37 degrees in water bath overnight.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">RE Digest Recipe for RFP-F,R</span><span class="c0">r</span></p><p class="c1"><span class="c0">40 uL RFP-F,R (based on the nanospec results above) [Edit 9/17/2010, 110 ul not there, reduced to 40]</span></p><p class="c1"><span class="c0">1 uL SpeI (edited changed from notI to speI )</span></p><p class="c1"><span class="c0">1 uL NotI</span></p><p class="c1"><span class="c0">5 uL 10X buffer for EcoRI [Edit, only EcoRI buffer]</span></p><p class="c1"><span class="c0">1 uL10x BSA (based on this specfic RE mix-- BSA is required)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Place into an eppendorf tube, pipette gently to mix. Incubate at 37 degrees in water bath overnight.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Results</span></p><p class="c1"><img height="366.0" src="images/image8.png" width="490.0"></p><p class="c1"><span class="c0 c12">Gel of multiple PCR reactions</span></p><p class="c1"><span class="c0 c12">100 bp Ladder| Aox1a FR| Aox1a FR2| Aox1b FR| Aox1b FR2| HybB FR| Ompa FR| RFP FR| RFP F2R| 100 bp ladder|</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">For Future</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">We have a strategy for each construct, written on the board. We must digest each PCR product to build our building blocks, then construct each construct through a series of ligations, digest, PCRs etc (See 9.18.2010 for strategy)</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9.18.2010</span></p><p class="c1"><span class="c0 c2">Goals</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">PCR purify the hybB F,R a</span><span class="c0 c5">nd RFP F,R</span><span class="c0"> (rxns from 9/16/2010)</span></li><li class="c3"><span class="c0 c5">Ligate hybB F,R and RFP F,R</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Notes:</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">RFP F,R was accidently digested with NotI and not SpeI.</span></li><li class="c3"><span class="c0">What to do? We will need to add 1 uL of SpeI and run overnight, so that the SpeI site is cleaved. <br>Just in case we need this RFP, we PCR-purified it today and stored in freezer, clearly labeled PARTLY DIGESTED RFP. If we need to use this RFP, it will need to be digested by SpeI.</span></li><li class="c3"><span class="c0">RFP band on the gel pic from 9/17/2010 was faint and nanospec showed low conc (8.8 ng /uL) versus the higher yields of the other rxns. </span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Ran PCR purification on HybB, stored in freezer. This HybB was nanospec&rsquo;ed on Friday and had a concentration of &nbsp;26.2 ng/uL. This has been digested by the right RE&rsquo;s, EcoRI and NotI, and ready to be ligated with RFP when it has been appropriately digested.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR Purificatio</span><span class="c0">n </span></p><p class="c1"><span class="c0">1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)</span></p><p class="c1"><span class="c0">2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).</span></p><p class="c1"><span class="c0">3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">4. Discarded flow-through. Placed the column back in the same tube.</span></p><p class="c1"><span class="c0">5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.</span></p><p class="c1"><span class="c0">7. Placed the column in a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">8. To elute DNA, 50 &nbsp;&micro;L autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2 c7">BASIC PLAN FOR BUILDING PLASMID CONSTRUCTS </span></p><p class="c1"><span class="c0">from the primered building blocks, which are stored in our freezer</span></p><p class="c1"><span class="c0">A) RUN GEL - 2 uL each - to check size - this step completed for all building blocks</span></p><p class="c1"><span class="c0">B) PCR PURIFY leftovers - this step completed for all building blocks</span></p><p class="c1"><span class="c0">C) NANOSPEC - record concentrations - this step completed for all building blocks</span></p><p class="c1"><span class="c0 c7">D) DIGEST</span><span class="c0"> with restriction enzymes, overnight</span></p><p class="c1"><span class="c0 c7">E) PCR PURIFY</span><span class="c0"> digestion products</span></p><p class="c1"><span class="c0 c7">F) LIGATION</span><span class="c0"> of gene building blocks - we are starting with HybB &amp; RFP</span></p><p class="c1"><span class="c0 c7">G) PCR </span><span class="c0">ligation products, HybB-RFP</span></p><p class="c1"><span class="c0 c7">H) DIGEST</span><span class="c0"> products, HybB-RFP</span></p><p class="c1"><span class="c0 c7">I) RUN GEL</span><span class="c0"> on small amount of digestion products, HybB-RFP</span></p><p class="c1"><span class="c0 c7">J) PCR PURIFY</span><span class="c0"> the rest of the digestion products, HybB-RFP</span></p><p class="c1"><span class="c0 c7">K) DIGEST</span><span class="c0"> the vector - we are using pBS1A3</span></p><p class="c1"><span class="c0 c7">L) PCR PURIFY</span><span class="c0"> the vector</span></p><p class="c1"><span class="c0 c7">M) LIGATION </span><span class="c0">of gene with vector, HybB-RFP and pBS1A3</span></p><p class="c1"><span class="c0 c7">N) TRANSFORMATION </span><span class="c0">of plasmid into E. coli, run overnight</span></p><p class="c1"><span class="c0 c2">Note- </span><span class="c0"> Do a nanospec after PCR purifications</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">NOTES: </span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Steps in BOLD are going to be repeated with each plasmid construct. Steps A,B, and C have already been done for each building block and won&rsquo;t be repeated unless there is a specific issue with a particular building block.</span></li><li class="c3"><span class="c0">Steps K and L do not necessarily need to wait until step J is completed to be run. They are listed this way so there is no confusion about what is being digested or PCR purified at each step. </span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Plan for this week:</span></p><p class="c1"><span class="c0">Weekend: PCR purify hybB, run gel for pSB1A3</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Mon: Redo PCR RFP; digest RFP (O/N)</span></li><li class="c3"><span class="c0">Tues: ligate hyB+RFP (O/N), Digest pSB1A3 (O/N)</span></li><li class="c3"><span class="c0">Wed: PCR ; digest (1hr); PCR purify (digests of hyb+rfp and pSB1a3) at same time we run gel to check results; ligate O/N</span></li><li class="c3"><span class="c0">Thurs: Tranformation of cells by hyb+RFP+psb1a3</span></li><li class="c3"><span class="c0">Fri: check plates</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/20/2010</span></p><p class="c1"><span class="c0 c2">Goals</span><span class="c0">: </span></p><ol class="c10"><li class="c3" value="1"><span class="c0 c22">Redo PCR of mRFP F,R; mRFP F2,R</span></li><li class="c3"><span class="c0 c33">Digest mRFP F,R; mRFP F2, R O/N (not possible until we get enzymes) </span></li><li class="c3"><span class="c0 c13">RUN GEL - 2 uL each - to check size - this step completed for all building blocks</span></li><li class="c3"><span class="c0 c13">PCR PURIFY leftovers - this step completed for all building blocks</span></li><li class="c3"><span class="c0 c13">NANOSPEC - record concentrations - this step completed for all building blocks</span></li><li class="c3"><span class="c0 c13">Transform novablue with mRFP that ryan gave us (labeled mRFP H) to create our own crytostock</span></li></ol><p class="c1"><span class="c0 c2">Notes:</span></p><ol class="c10"><li class="c3" value="1"><span class="c0 c7">Ryan gave us a fresh stock of mRFP plasmid</span><span class="c0">. </span><span class="c0 c2">We only get this one</span><span class="c0">! It is precious like gold or diamonds!</span></li><li class="c3"><span class="c0">Since the original mRFP has a Nde I site in the MCS, we are worried other products may run at the same size. To avoid this worry, we will use gel extraction to extract only our band of interest. </span></li></ol><p class="c1"><span class="c0 c2">mRFP</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">in the plasmid PET15B</span></li><li class="c3"><span class="c0">Use Novablue to create a cryostock. (transform, grow on plate, pick colony, grow in liquid media, take cryostock)</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Protocols</span></p><p class="c1"><span class="c0 c2">&nbsp;</span></p><p class="c1"><span class="c0">9am </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR Protocol </span><span class="c0"> for mRFP F,R; mRFP F2, R</span></p><p class="c1"><span class="c0">26.5 uL H2O</span></p><p class="c1"><span class="c0">10 uL </span><span class="c0 c7">PHUSION 5X Reaction buffer</span></p><p class="c1"><span class="c0">5 uL forward primer </span></p><p class="c1"><span class="c0">5 uL reverse primer</span></p><p class="c1"><span class="c0">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c1"><span class="c0 c7">2 uL</span><span class="c0"> template DNA</span></p><p class="c1"><span class="c0">0.5 uL polymerase enzyme, </span><span class="c0 c7">PHUSION</span></p><p class="c1"><span class="c0 c7">Total Volume= 50 uL</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">Ran PCR results (pre purification) on gel)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">12pm</span></p><p class="c1"><span class="c0 c2">Heat shock transformation of the plasmids into our bacteria</span></p><p class="c1"><span class="c0">10 &#1405;L Nova Blue cells + 5 &#1405;L of MRFP plasmid (from stock that Ryan gave us this morning).</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. Left cells and ligation reaction products on ice.</span></p><p class="c1"><span class="c0">2. Added plasmid (5&#1405;L) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).</span></p><p class="c1"><span class="c0">3. Left cells on ice for 30 min. </span></p><p class="c1"><span class="c0">4. Applied heat shock of 45 seconds in 42C bath.</span></p><p class="c1"><span class="c0">5. Put tubes on ice for 2 min. </span></p><p class="c1"><span class="c0">6. Added 250 &#1405;L of LB (room temp.)</span></p><p class="c1"><span class="c0">7. Incubated 1 hour at 37 C</span></p><p class="c1"><span class="c0">8. Plated 100&#1405;L and left plate in the 37 degrees incubator &nbsp;</span></p><p class="c1"><span class="c0">9. Incubated overnight at 37C. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Results</span></p><ol class="c29"><li class="c3" value="1"><span class="c0">Gel picture of mRFP FR, F2R (Insert gel pic!)</span></li></ol><ol class="c34"><li class="c14" value="1"><span class="c0">The bands of both PCRs (pre digest) were between approximately around &nbsp;700-800 </span></li></ol><p class="c1"><img height="288.0" src="images/image3.png" width="385.0"></p><p class="c1"><span class="c0 c12">Gel of mRFP FR and mRFP F2R</span></p><p class="c1"><span class="c0 c12">100bp ladder|mRFP FR|mRFP F2R|Control</span></p><p class="c1"><span class="c0 c2">PCR Purificatio</span><span class="c0">n-mRFP</span></p><p class="c1"><span class="c0">1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)</span></p><p class="c1"><span class="c0">2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).</span></p><p class="c1"><span class="c0">3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">4. Discarded flow-through. Placed the column back in the same tube.</span></p><p class="c1"><span class="c0">5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.</span></p><p class="c1"><span class="c0">7. Placed the column in a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">8. To elute DNA, 30 &nbsp;&micro;L autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.</span></p><p class="c1"><span class="c0 c2">&nbsp;</span></p><p class="c1"><span class="c0 c2">Nanospec Results</span></p><p class="c1"><span class="c0">mRFP, F: 137.2ng/uL</span></p><p class="c1"><span class="c0">mRFP, F2: 132.3 ng/uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">For Future</span></p><ol class="c10"><li class="c3" value="1"><span class="c0 c5">Digest first thing Tuesday!</span></li><li class="c3"><span class="c0">Richard suggested getting two squirt bottles- 1 for water, 1 for bleach (to kill cells)</span></li><li class="c3"><span class="c0">Check if transformation of Nova Blue with mRFP worked!</span></li></ol><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/21/2010</span></p><p class="c1"><span class="c0">Results from 9/20/2010</span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transformation of mRFP in NB cells successful (both plates). </span></p><p class="c1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></p><p class="c1"><span class="c0 c2">Starter Cultures</span></p><p class="c1"><span class="c0">Make starter cultures from both mRFP:NB plates, allow to grow overnight. </span></p><p class="c16"><span class="c0">3mL LB + 3uL 1000X CARB</span></p><p class="c16"><span class="c0">put in 37C incubator with shaking overnight</span></p><p class="c16"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Received shipment of SpeI (200 uL) and XmaI (50 uL) from Promega, put in -20C freezer (Gaucher Lab).</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">For 9/22/2010:</span></p><ol class="c4"><li class="c14" value="1"><span class="c0">Begin digests 9am, do 3 hours insteadf of overnight, and ligation in evening</span></li><li class="c14"><span class="c0">create cryostocks from starter cultures grown on 9/21/2010.</span></li><li class="c14"><span class="c0">Couldn&rsquo;t start RE digest of RFP today- could not find RE&rsquo;s. Will ask Megan Wednesday morning.</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/22/2010- (Mitesh, Scott, Gita)</span></p><p class="c1"><span class="c0 c2">Goals</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Digest of RFP F,R</span></li><li class="c3"><span class="c0">PCR purify the digest</span></li><li class="c3"><span class="c0">Ligate hyBB FR, to RFP F,R</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Notes: made a stock of 1 ug/ul BSA for easier RE digests</span></p><p class="c1"><span class="c0">I am following the suggested RE protocol listed on the Promega literature that came with the RE&rsquo;s</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">RE Digest Recipe for RFP-F,R</span><span class="c0">r</span></p><p class="c1"><span class="c0">4.5 uL H20</span></p><p class="c1"><span class="c0">2uL 10X Promega Buffer D</span></p><p class="c1"><span class="c0">10 uL RFP-F,R ( from 9.20.2010, 137 ug/ul)</span></p><p class="c1"><span class="c0">2 uL BSA (0ug/uL, to a final conc of .1mg/ml)</span></p><p class="c1"><span class="c0">0.75 uL SpeI (edited changed from notI to speI )</span></p><p class="c1"><span class="c0">0.75 uL NotI</span></p><p class="c1"><span class="c0 c7">Total=20 ul total </span></p><p class="c1"><span class="c0">Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)</span></p><p class="c1"><span class="c0">Start time is 10:35 am</span></p><p class="c1"><span class="c0">End time should be 1:35 pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Purification of RFP digest</span></p><p class="c1"><span class="c0">1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)</span></p><p class="c1"><span class="c0">2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).</span></p><p class="c1"><span class="c0">3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">4. Discarded flow-through. Placed the column back in the same tube.</span></p><p class="c1"><span class="c0">5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.</span></p><p class="c1"><span class="c0">7. Placed the column in a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">8. To elute DNA, 30 &nbsp;&micro;L autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.</span></p><p class="c1"><span class="c0 c2">&nbsp;</span></p><p class="c1"><span class="c0 c2">Nanospec</span></p><p class="c1"><span class="c0">RFP Digest (purified)- 41 ng/uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Ligation of RFP-FR to HybB-FR</span></p><p class="c1"><span class="c0">using hybB from 9/18/2010 and RFP from 9/21/2010</span></p><p class="c1"><span class="c0 c12">Calculating equivalents:</span></p><p class="c1"><span class="c0">RFP- [41 ng/uL]/678 bp= 0.06 eq/uL</span></p><p class="c1"><span class="c0">hyBb-[26 ng/uL]/393 bp = 0.06 eq/uL</span></p><p class="c1"><span class="c0">for linear ligations, use a 1:1 ratio of products</span></p><p class="c1"><span class="c0 c2">Ligation:</span></p><p class="c1"><span class="c0">2 uL of RFP FR (SpeI, NotI digested; purified)</span></p><p class="c1"><span class="c0">2 uL of HybB FR (EcoRI, NotI digested; purified)</span></p><p class="c1"><span class="c0">1 uL 10x Ligase Buffer</span></p><p class="c1"><span class="c0">4.5 uL H20</span></p><p class="c1"><span class="c0">0.5 uL T4 Ligase</span></p><p class="c1"><span class="c0 c7">Total=10 uL</span></p><p class="c1"><span class="c0">Leave RT for 1 hour</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Start time: 3:20 pm</span></p><p class="c1"><span class="c0">End time: 4:20 pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR of hybB+Mrfp construct </span></p><p class="c1"><span class="c0">3 uL of ligation reaction</span></p><p class="c1"><span class="c0">25.5 uL H2O</span></p><p class="c1"><span class="c0">10 uL </span><span class="c0 c7">PHUSION 5X Reaction buffer</span></p><p class="c1"><span class="c0">5 uL forward primer </span></p><p class="c1"><span class="c0">5 uL reverse primer</span></p><p class="c1"><span class="c0">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c1"><span class="c0">0.5 uL polymerase enzyme, </span><span class="c0 c7">PHUSION</span></p><p class="c1"><span class="c0 c7">Total Volume= 50 uL</span></p><p class="c1"><span class="c0">Start: 5pm</span></p><p class="c1"><span class="c0">End: Thursday</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">For Future</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Take out pcr of mrfp+hybB</span></li><li class="c3"><span class="c0">Run 2 uL on gel, along with just the RFP-FR and hybB-FR (1hr)</span></li><li class="c3"><span class="c0">Digest the construct, along with the vector psb1a3 at the same time (3 hrs)</span></li><li class="c3"><span class="c0">Ligate the construct to psb1a3 (1 hr)</span></li><li class="c3"><span class="c0">Transform into e coli (1.5 hrs)</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9.23.2010 (Scott, Rob, Gita)</span></p><p class="c1"><span class="c0 c2">Goals</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Run PCR of RFP+hyBb on gel</span></li><li class="c3"><span class="c0">Digest the construct, along with vecotr psb1a3</span></li><li class="c3"><span class="c0">ligate the construct to psb1a3</span></li><li class="c3"><span class="c0">transform into e. coli</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Notes:</span></p><p class="c1"><span class="c0">Predicted size of hyb+MRFP~1000bp</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Protocols</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Running PCR product on gel</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">The pcr was left overnight at 12 c, and I am running 4 uL on a gel</span></li><li class="c3"><span class="c0">4 &nbsp;uL sample + 1 uL running dye</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Notes:</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">I could not see any stained DNA, even the ladder. This means the EtBr degraded in the gels. Solution, suggested by Richard, is to keep a bottle of 1% Agarose/TBE solution at RT. This solution represents the first step of making a gel. Then whenever we need a gel, we start from the pre-made solution, add Etbr, and procede as normal. Start with fresh gels.</span></li><li class="c3"><span class="c0">PCR products store in yellow box</span></li><li class="c3"><span class="c0">Building blocks in blue rack</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Making gel for PCR</span></p><p class="c1"><span class="c0">1. &nbsp;Heat 30 mL of 1% agarose solution &nbsp;in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don&rsquo;t vaporize it, especially near your face!</span></p><p class="c1"><span class="c0">2. Add 35 &#1405;L EtBr (edited from 45 uL, make 1000X)</span></p><p class="c1"><span class="c0">3. Pour gel and allow to harden. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Results</span></p><p class="c1"><img height="313.0" src="images/image1.png" width="419.0"></p><p class="c1"><span class="c0 c12">Gel from 9.23.2010.</span></p><p class="c1"><span class="c0 c12">100 bp ladder| RFP-FR +HybB PCR|</span></p><p class="c1"><span class="c0 c12">Predicted Size- 1070 bp</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Due to multiple bands, we decided to gel extract the construct RFP+hyBB. To do this, we borrowed a larger Gel try and apparatus from the Hammer Lab (they had a box of things they were not using). The larger gel is approximately 80 ml and is larger in area. The new gel will help make cutting the band easier. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Making gel for PCR (Hammer Lab Apparatus)</span></p><p class="c1"><span class="c0">1. &nbsp;Heat 90 mL of 1% agarose solution &nbsp;in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don&rsquo;t vaporize it, especially near your face!</span></p><p class="c1"><span class="c0">2. Add 90 &#1405;L EtBr (edited from 45 uL, make 1000X)</span></p><p class="c1"><span class="c0">3. Pour gel and allow to harden. </span></p><p class="c1"><span class="c0">(I did 100 mL, but found the actual volume to be around 80 to 90 mL)</span></p><p class="c1"><span class="c0">Start: 4:10 pm</span></p><p class="c1"><span class="c0">End: 5:10 pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Gel Extraction Protocol</span></p><p class="c1"><span class="c0">1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.</span></p><p class="c1"><span class="c0">2. Weighed the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 &mu;L).</span></p><p class="c1"><span class="c0">3. Incubated at 50&ordm;C for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 &ndash; 3 min during the incubation.</span></p><p class="c1"><span class="c0">4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).</span></p><p class="c1"><span class="c0">5. Added 1 gel volume of isopropanol to the sample and mixed.</span></p><p class="c1"><span class="c0 c5">6. Placed a QIAquick spin column in a provided 2 mL collection tube.</span></p><p class="c1"><span class="c0">7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.</span></p><p class="c1"><span class="c0">8. Discarded flow-through and placed QIAquick column back in the same collection tube.</span></p><p class="c1"><span class="c0 c5">9. Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.</span></p><p class="c1"><span class="c0">10. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.</span></p><p class="c1"><span class="c0">11. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).</span></p><p class="c1"><span class="c0">12. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">13. To elute DNA, added 30 &nbsp;&mu;L water (pH 7.0 &ndash; 8.5), let the column stand for 1 min, and then centrifuged for 1 min.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><img height="394.0" src="images/image5.png" width="526.0"></p><p class="c1"><span class="c0 c12">Gel of RFP-FR + HybB FR PCR product (Before Gel Extraction)</span></p><p class="c1"><span class="c0 c12">30 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c1"><span class="c0 c12">Band to be excised is the 1100 bp band (2nd bright band from bottom)</span></p><p class="c1"><span class="c0 c12">&nbsp;</span></p><p class="c1"><img height="344.0" src="images/image7.png" width="460.0"></p><p class="c1"><span class="c0 c12">Gel of RFP-FR + HybB FR PCR product (After Gel Extraction)</span></p><p class="c1"><span class="c0 c12">30 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c1"><span class="c0 c12">&nbsp;</span></p><p class="c1"><span class="c0 c2">For Future-</span></p><p class="c1"><span class="c0 c12">Friday-</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">nanospec the gel extracted hyb+RFP construct (in blue box)</span></li><li class="c3"><span class="c0">Run 2 uL on gel, see if 1100 bp is confirmed</span></li><li class="c3"><span class="c0">if so, continue with strategy. </span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/24/2010</span><span class="c0 c2">Goals</span></p><ol class=""><li class="c3" value="4"><span class="c0 c43">nanospec the gel extracted hyb+RFP construct (in blue box)</span></li><li class="c3"><span class="c0 c43">Run 2 uL on gel, see if 1100 bp is confirmed</span></li><li class="c3"><span class="c0 c13">if so, digest the construct and vector psb1a3 (3 hrs); ligate (1hr), transform (1.5 hours)</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Protocols</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">1 % Agarose Gel</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">I found that the small gel trays need only 25-30 mL of solution (less than 30)</span></li><li class="c3"><span class="c0">I heated 30 mL of 1 % Agarose solution for approximately 45 seconds. I then added 30 uL of EtBr (use blue gloves) and poured into the prepared gel tray (tray inside of assembly, with combs). Cool for 1 hr. The gel will be done when there is a faint blue hue visible when looking at it. </span></li><li class="c3"><span class="c0">Gel Start: 9:10 am</span></li><li class="c3"><span class="c0">End 10:10 am</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Nanospec of Gel Extract from RFP+HybB PCR </span></p><p class="c1"><span class="c0">19.2 ng/uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">(inset gel pic from etbr lab)</span></p><p class="c1"><span class="c0 c12">Gel of Excised 1100 bp band of &nbsp;RFP-FR + HybB FR PCR product </span></p><p class="c1"><span class="c0 c12">4 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c1"><span class="c0 c12">Band to be excised is the 1100 bp band (2nd bright band from bottom)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">RE Digest Recipe for HybB +RFP PCR (Mitesh did this today)</span></p><p class="c1"><span class="c0">3.5 uL H20</span></p><p class="c1"><span class="c0">3uL 10X Promega Buffer E o</span><span class="c0 c5">r Multicore</span></p><p class="c1"><span class="c0">20uL HybB+RFP (excised 1100 bp badn from PCR) ( from 9.23.2010, 19 ng/ul)</span></p><p class="c1"><span class="c0">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c1"><span class="c0">0.75 uL SpeI </span></p><p class="c1"><span class="c0">0.75 uL EcoRI</span></p><p class="c1"><span class="c0 c7">Total=30 ul total </span></p><p class="c1"><span class="c0">Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">RE Digest Recipe for pSB1A3 (Debika did this today)</span></p><p class="c1"><span class="c0">3.5 uL H20 (edit 9/24/2010- should be 8.5 uL)</span></p><p class="c1"><span class="c0">5uL 10X Promega Buffer E </span><span class="c0 c5">or Multicore</span></p><p class="c1"><span class="c0">10 uL pSB1A3 (9.16.2010, 100 ng/uL)</span></p><p class="c1"><span class="c0">5 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c1"><span class="c0">0.75 uL SpeI </span></p><p class="c1"><span class="c0">0.75 uL EcoRI</span></p><p class="c1"><span class="c0 c7">Total=50 ul total </span></p><p class="c1"><span class="c0">Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR Purfiy the two digests (RFP+Hyb construct, pSB1A3)</span></p><p class="c1"><span class="c0">Note- We have had the best results when eluting in </span><span class="c0 c7">30 uL of H20</span></p><p class="c1"><span class="c0 c2">&nbsp;</span></p><p class="c1"><span class="c0 c2">Nanospec</span></p><p class="c1"><span class="c0">pSB1A3 Digest (purified) = 6.2</span><span class="c0 c27"> </span><span class="c0">ng/uL</span></p><p class="c1"><span class="c0">RFP+HYBB Digest (purified)= 4.4ng/uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Ligation of pSB1A3 to [RFP-FR+ HybB-FR]</span><span class="c0"> (from 9.23.2010)</span></p><p class="c1"><span class="c0 c12">Calculating equivalents:</span></p><p class="c1"><span class="c0">pSB1A3- [ </span><span class="c0 c27">6.2</span><span class="c0"> ng/uL]/ 2100 bp= &nbsp;3x10^-3 eq/uL</span></p><p class="c1"><span class="c0">hyBb+RFP-[ </span><span class="c0 c27">4.4</span><span class="c0"> ng/uL]/1100 bp = 4x10^-3 &nbsp;eq/uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">For plasmid to linear ligations, use a 1:2 ratio of vector:products if the products are purified (1:4 if not)</span></li></ol><ol class="c4"><li class="c14" value="1"><span class="c0">E.g. 1 equivalent of vector to 2 equivalent of linear product</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Ligation Protocol:</span></p><p class="c1"><span class="c0"> 3 uL of RFP + HYB (~ 2 to 3 uL)</span></p><p class="c1"><span class="c0"> 2 uL of &nbsp;Plasmid pSB1A3 (~ 1/2x amount of linear product on eq/uL basis)</span></p><p class="c1"><span class="c0">1 uL 10x Ligase Buffer</span></p><p class="c1"><span class="c0">3.5 uL H20</span></p><p class="c1"><span class="c0">0.5 uL T4 Ligase (enzyme-- keep in freezer-- add last of all)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">Total=10 uL</span></p><p class="c1"><span class="c0">Leave RT for 1 hour</span></p><p class="c1"><span class="c0">Start: 5:23 pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Transformation</span></p><p class="c1"><span class="c0 c2">Heat shock transformation of the plasmids into our bacteria</span></p><p class="c1"><span class="c0">10 &#1405;L Nova Blue cells + 5 &#1405;L of Ligation Reaction</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. Left cells and ligation reaction products on ice.</span></p><p class="c1"><span class="c0">2. Added plasmid (5&#1405;L) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).</span></p><p class="c1"><span class="c0">3. Left cells on ice for 30 min. </span></p><p class="c1"><span class="c0">4. Applied heat shock of 45 seconds in 42C bath.</span></p><p class="c1"><span class="c0">5. Put tubes on ice for 2 min. </span></p><p class="c1"><span class="c0">6. Added 250 &#1405;L of LB (room temp.)</span></p><p class="c1"><span class="c0">7. Incubated 1 hour at 37 C</span></p><p class="c1"><span class="c0">8. Plated 100&#1405;L (x2 plates) and left plates in the 37 degrees incubator &nbsp;</span></p><p class="c1"><span class="c0">9. Incubated overnight at 37C. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Notes for Future-</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Aliquots of BL21 and Novablue are in our -70c freezer</span></li><li class="c3"><span class="c0">Autoclave fresh pipette tips- there is confusion over which tips fit what pipettes- we need to calibrate to make sure our tips and pipettes are giving us the volumes we want</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">SEPTEMBER 25, 2010</span></p><p class="c1"><span class="c0 c2">Goals-</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Begin digesting the building blocks necessary to create the rest of the constructs</span></li><li class="c3"><span class="c0">Pick colonies from the pSB1A3+Hyb+RFP transformation and grow overnight</span></li></ol><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">Nanospec results </span><span class="c0">(From tubes dated 9/17/2010)</span></p><p class="c1"><span class="c0">AOX1a-F,R = 99.3 ng/uL</span></p><p class="c1"><span class="c0">AOX1a-F,R2 = 50.0 ng/uL</span></p><p class="c1"><span class="c0">AOX1b-F,R = 48.5 ng/uL</span></p><p class="c1"><span class="c0">AOX1b-F,R2 = 42.1 ng/uL</span></p><p class="c1"><span class="c0">OmpA-F,R = 27.3 ng/uL</span></p><p class="c1"><span class="c0">RFP-F2,R = 10.8 ng/uL</span></p><p class="c1"><span class="c0">Hyb-F,R = 26.2 ng/uL</span></p><p class="c1"><span class="c0">RFP-F,R = 8.8 ng/uL</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">RE Double Digest Recipe for RFP-F2,R</span></p><p class="c1"><span class="c0">0 uL H20 (plenty in the RFP-F2,R tube, which is very dilute)</span></p><p class="c1"><span class="c0">5 uL 10X Promega Buffer D</span></p><p class="c1"><span class="c0">40 uL RFP-F2,R ( from 9.17.2010, 10.8 ng/ul)</span></p><p class="c1"><span class="c0">5 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c1"><span class="c0">0.75 uL SpeI </span></p><p class="c1"><span class="c0">0.75 uL NdeI</span></p><p class="c1"><span class="c0 c7">Total=51.5 ul total </span></p><p class="c1"><span class="c0">Start :4pm</span></p><p class="c1"><span class="c0">End: 7 pm</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">RE Double Digest Recipe for OmpA F,R</span></p><p class="c1"><span class="c0">1.5 uL H20 </span></p><p class="c1"><span class="c0">5 uL 10X Promega Multi-Core Buffer</span></p><p class="c1"><span class="c0">37 uL OmpA-F,R ( from 9.17.2010, 27.3 ng/ul)</span></p><p class="c1"><span class="c0">5 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c1"><span class="c0">0.75 uL NotI </span></p><p class="c1"><span class="c0">0.75 uL XmaI</span></p><p class="c1"><span class="c0 c7">Total=50 ul total </span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c7">Went in at 2:55 pm</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">RE Double Digest Recipe for AOX1a F,R</span></p><p class="c1"><span class="c0">12.5 uL H20 </span></p><p class="c1"><span class="c0">3 uL 10X Promega Buffer B</span></p><p class="c1"><span class="c0">10 uL AOX1a-F,R ( from 9.17.2010, 99.3 ng/ul)</span></p><p class="c1"><span class="c0">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c1"><span class="c0">0.75 uL SpeI </span></p><p class="c1"><span class="c0">0.75 uL XmaI</span></p><p class="c1"><span class="c0 c7">Total=30 ul total </span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c7">Went in at 3:05 pm</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">RE Double Digest Recipe for AOX1a F,R2</span></p><p class="c1"><span class="c0">2.5 uL H20 </span></p><p class="c1"><span class="c0">3 uL 10X Promega Multi Core Buffer</span></p><p class="c1"><span class="c0">20 uL AOX1a-F,R2 ( from 9.17.2010, 50 ng/ul)</span></p><p class="c1"><span class="c0">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c1"><span class="c0">0.75 uL NdeI </span></p><p class="c1"><span class="c0">0.75 uL XmaI</span></p><p class="c1"><span class="c0 c7">Total=30 ul total </span></p><p class="c1"><span class="c0">Start 4 pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">RE Double Digest Recipe for AOX1b F,R</span></p><p class="c1"><span class="c0">2.5 uL H20 </span></p><p class="c1"><span class="c0">3 uL 10X Promega Buffer B</span></p><p class="c1"><span class="c0">20 uL AOX1b-F,R ( from 9.17.2010, 48.5 ng/ul)</span></p><p class="c1"><span class="c0">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c1"><span class="c0">0.75 uL SpeI </span></p><p class="c1"><span class="c0">0.75 uL XmaI</span></p><p class="c1"><span class="c0 c7">Total=30 ul total </span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c7">Went in at 3:10 pm</span></p><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><span class="c0 c2">RE Double Digest Recipe for AOX1b F,R2</span></p><p class="c1"><span class="c0">0 uL H20 </span></p><p class="c1"><span class="c0">3 uL 10X Promega Multi Core Buffer </span></p><p class="c1"><span class="c0">22.5 uL AOX1b-F,R ( from 9.17.2010, 42.1 ng/ul)</span></p><p class="c1"><span class="c0">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c1"><span class="c0">0.75 uL NdeI </span></p><p class="c1"><span class="c0">0.75 uL XmaI</span></p><p class="c1"><span class="c0 c7">Total=30 ul total </span></p><p class="c1"><span class="c0">Start 4 pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)</span></p><p class="c1"><span class="c0">Start time is </span><span class="c0 c27">3, 4</span><span class="c0">pm (two sets of reactions, noted in the individual descriptions)</span></p><p class="c1"><span class="c0">End time should be </span><span class="c0 c27">6,7 </span><span class="c0">pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Preparing liquid culture of pSB1A3+hybB+RFP</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">3 mL of LB + 3 uL of Carb into a culture tube. Picked 2 colonies from plates from 9.24.2010</span></li><li class="c3"><span class="c0">O/N at 37c in our incubator-shaker</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">For Future-</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">PCR purify the digests</span></li><li class="c3"><span class="c0">start first round of ligations</span></li><li class="c3"><span class="c0">PCR the ligation products</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/27/2010</span></p><p class="c1"><span class="c0 c2">Goals</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">PCR purify digests of building blocks from 9/25/2010</span></li><li class="c3"><span class="c0">Start first round of ligations for each construct</span></li><li class="c3"><span class="c0">Check ligations on gel</span></li><li class="c3"><span class="c0">Digest; troubleshoot ligations if necessary</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Protocols</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR Purification of Digests from 9/25/2010</span><span class="c0"> (Ompa FR , AOX1a/b FR and FR2, RFP F2R)</span></p><p class="c1"><span class="c0">Reactions I am purifying:</span></p><p class="c1"><span class="c0">RFP-F2R (NdeI, SpeI)</span></p><p class="c1"><span class="c0">OmpA FR (NotI, XmaI)</span></p><p class="c1"><span class="c0">Aox1a-FR (Xma, SpeI)</span></p><p class="c1"><span class="c0">Aox1a-FR2 (Xma, NdeI)</span></p><p class="c1"><span class="c0">Aox1b-FR (Xma, SpeI)</span></p><p class="c1"><span class="c0">Aox1b-FR2 (Xma, NdeI)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">1. Added 5 volumes of Buffer PBI to 1 volume of the sample and mix (in a clean 1.5 mL eppendorf)</span></p><p class="c1"><span class="c0">2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).</span></p><p class="c1"><span class="c0">3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">4. Discarded flow-through. Placed the column back in the same tube.</span></p><p class="c1"><span class="c0">5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 &ndash; 60 secs.</span></p><p class="c1"><span class="c0">6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.</span></p><p class="c1"><span class="c0">7. Placed the column in a clean 1.5 mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">8. To elute DNA, 30 &nbsp;&micro;L autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Nanospec of Purified digests</span></p><p class="c1"><span class="c0">RFP-F2R (NdeI, SpeI)= 28.6 ng/uL</span></p><p class="c1"><span class="c0">OmpA FR (NotI, XmaI)= 15 ng/uL</span></p><p class="c1"><span class="c0">Aox1a-FR (Xma, SpeI)= 12 ng/uL</span></p><p class="c1"><span class="c0">Aox1a-FR2 (Xma, NdeI)= 25 ng/uL</span></p><p class="c1"><span class="c0">Aox1b-FR (Xma, SpeI)= 23 ng/uL</span></p><p class="c1"><span class="c0">Aox1b-FR2 (Xma, NdeI)= 21.3 ng/uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Ligations</span></p><p class="c1"><span class="c0">hyb= [26.2 ng/ul ]/ 393 bp = 0.067 eq/uL</span></p><p class="c1"><span class="c0">ompa = [15 ng/uL]/81 bp = 0.185 eq/uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Ligation of HyBb+ Ompa</span></p><p class="c1"><span class="c0">5.7 uL H20</span></p><p class="c1"><span class="c0"> 2 uL of HybB</span></p><p class="c1"><span class="c0"> 0.8 &nbsp;uL of Ompa </span></p><p class="c1"><span class="c0">1 uL 10x Ligase Buffer</span></p><p class="c1"><span class="c0">0.5 uL T4 Ligase (enzyme-- keep in freezer-- add last of all)</span></p><p class="c1"><span class="c0 c12">Total 10 uL</span></p><p class="c1"><span class="c0">RT for 1 hr. - Start time: 11:25 am</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Aliquot T4 Buffer:</span></p><p class="c1"><span class="c0">Make aliquots (5 uL) of the T4 Ligase Buffer so as to not continuously repeat freeze-thaw cycles. </span></p><p class="c1"><span class="c0">USE FROM ALIQUOTS FROM NOW ON - NOT THE GREEN-CAPPED EPPENDORF.</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Miniprep of pSB1A3+hybB+ompA</span></p><p class="c1"><span class="c0">1. Remove inoculation tubes from inoculation (37C shaker).</span></p><p class="c1"><span class="c0">2. Obtain P1 buffer from 4C refrigerator.</span></p><p class="c1"><span class="c0">3. Take centrifuge tubes and add 1.5mL of inoculated cells. </span></p><p class="c1"><span class="c0">4. Centrifuge at 3000 rpm (low) for 1-2 min. </span></p><p class="c1"><span class="c0">5. Spin until white pellet of cells forms at the bottom and liquid is more clear. </span></p><p class="c1"><span class="c0">6. Take off supernatant and discard. </span></p><p class="c1"><span class="c0">7. Repeat steps 4-6. </span></p><p class="c1"><span class="c0">8. Resuspend pelleted bacterial cells in 250&#1405;L P1 buffer. </span></p><p class="c1"><span class="c0">9. &nbsp;Add 250&#1405;L P2 buffer and invert 4-6 times (DO NOT VORTEX - doing so will shear DNA!)</span></p><p class="c1"><span class="c0">10. Add 350&#1405;L buffer N3 and immediately invert 4-6 times. </span></p><p class="c1"><span class="c0">11. Centrifuge for 10 min. at 13,000 rpm. </span></p><p class="c1"><span class="c0">12. Take supernatant and add to spin columns. </span></p><p class="c1"><span class="c0">13. Spin 30-60 sec. and discard flow through. </span></p><p class="c1"><span class="c0">14. Wash column with 750&#1405;L buffer PE and centrifuge 1 min. </span></p><p class="c1"><span class="c0">15. Discard flow through and centrifuge and additional minute.</span></p><p class="c1"><span class="c0">16. Please column into a clean 1.5mL microcentrifuge tube.</span></p><p class="c1"><span class="c0">17. Elute DNA by adding 30&#1405;L dH2O. </span></p><p class="c1"><span class="c0">18. Let stand for 1 min., then centrifuge for 1 min. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Making a 1 % gel </span></p><p class="c1"><span class="c0">25*4=100 mL</span></p><p class="c1"><span class="c0">We found that we had extra left over. </span></p><p class="c1"><span class="c0">Start at 1:13 pm</span></p><p class="c1"><span class="c0">End: 2:13 pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">PCR of Hyb+OmpA ligation </span></p><p class="c1"><span class="c0">3 uL of product of ligation reaction</span></p><p class="c1"><span class="c0">25.5 uL H2O</span></p><p class="c1"><span class="c0">10 uL </span><span class="c0 c7">PHUSION 5X Reaction buffer</span></p><p class="c1"><span class="c0">5 uL HybB-F forward primer </span></p><p class="c1"><span class="c0">5 uL OmpA-R reverse primer</span></p><p class="c1"><span class="c0">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c1"><span class="c0">0.5 uL polymerase enzyme, </span><span class="c0 c7">PHUSION</span></p><p class="c1"><span class="c0 c7">Total Volume= 50 uL</span></p><p class="c1"><span class="c0">Start: 12:57 pm</span></p><p class="c1"><span class="c0">End: 3:00 pm (approx)</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Plan:</span></p><p class="c1"><span class="c0">Once PCR is finished, run 4 uL of the reaction on the gel. Check for a band running at a size of 393+81=474 bp, so approximately mid 400-500 bp. If band is observed, prepare for the digest (check for the RE sites based on what primers were used).</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Gel Picture</span></p><p class="c1"><span class="c0">insert gel pic from computer</span></p><p class="c1"><span class="c0">band around 500 bp. </span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Nanospec PCR</span></p><p class="c1"><span class="c0">Christina</span></p><p class="c1"><span class="c0">326 ng/uL</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/28/2010</span></p><p class="c1"><span class="c0 c2">Goals</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">The gel picture of the hybB+ompa is in the folder. </span></li></ol><ol class="c4"><li class="c14" value="1"><span class="c0">The band is bright at 500 bp, and our predictions confirm the results. </span></li><li class="c14"><span class="c0">PCR purify the PCR (in the yellow box)</span></li><li class="c14"><span class="c0">Nanospec</span></li></ol><p class="c1"><span class="c0 c2">Notes</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">Christina+Rob PCR purified the hyb+ompa PCR.</span></li><li class="c3"><span class="c0">Richard suggested we can PCR Aox+RFP, then pcr that to the hyb+ompa. This wil take care of two constructs. The other two require Aox a/b to be attached to the hybb+ompa construct</span></li><li class="c3"><span class="c0">For the constructs that have Aox+RFP, we can do a ligation of Aox to RFP; then, PCR that part and ligate it to the Hyb+ompa.</span></li></ol><ol class="c34"><li class="c3" value="1"><span class="c0">For the AOX-RFP constructs:</span></li></ol><ol class="c32"><li class="c14" value="1"><span class="c0">Ligate Aox1a-FR2 to RFP-F2R</span></li><li class="c14"><span class="c0">LigateAox1b-FR2 to RFP-F2R</span></li><li class="c14"><span class="c0">PCR each ligation reaction</span></li><li class="c14"><span class="c0">PCR purify</span></li><li class="c14"><span class="c0">Check results on gel</span></li></ol><ol class=""><li class="c46" value="1"><span class="c0">Gel extract if PCR results in multiple bands</span></li></ol><ol class="c32"><li class="c14" value="6"><span class="c0">Digest the Aox-RFP construct</span></li><li class="c14"><span class="c0">PCR purify</span></li><li class="c14"><span class="c0">Ligate the Aox-RFP digests to Hyb-Ompa digest</span></li><li class="c14"><span class="c0">PCR the entire constructs</span></li></ol><ol class="c34"><li class="c3" value="2"><span class="c0">For the HybB-Ompa-Aox constructs:</span></li></ol><ol class="c32"><li class="c14" value="1"><span class="c0">Ligate the HybB-Ompa to Aox1a-FR or Aox1b-FR</span></li><li class="c14"><span class="c0">PCR the constructs</span></li><li class="c14"><span class="c0">PCR Purify</span></li><li class="c14"><span class="c0">Check results on gel</span></li></ol><ol class="c29"><li class="c46" value="1"><span class="c0">if multiple bands, gel extract</span></li></ol><ol class="c32"><li class="c14" value="5"><span class="c0">Ligate Hyb-Ompa-Aox to pSB1A3 </span></li><li class="c14"><span class="c0">Transform into cells or PCR entire vector</span></li></ol><p class="c1"><span class="c7 c44">Constructs</span></p><hr><p class="c1"><span class="c0 c7">&nbsp;</span></p><p class="c1"><img height="414.0" src="images/image6.png" width="709.0"><img height="443.0" src="images/image4.png" width="705.0"><img height="411.0" src="images/image0.png" width="706.0"></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><hr><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c7">9/29/2010</span></p><p class="c1"><span class="c0 c2">Goals</span></p><ol class="c29"><li class="c3" value="1"><span class="c0">Ligate Aox1a-FR2 to RFP-F2R (1hr)</span></li><li class="c3"><span class="c0">LigateAox1b-FR2 to RFP-F2R (1hr at same time as step 1)</span></li><li class="c3"><span class="c0">Digest HybB.Ompa (3 hours)</span></li></ol><ol class="c34"><li class="c14" value="3"><span class="c0">Ligate the HybB-Ompa to Aox1a-FR &nbsp;</span></li><li class="c14"><span class="c0">Ligate the HybB-Ompa to Aox1b-FR </span></li></ol><ol class=""><li class="c3" value="4"><span class="c0">PCR each ligation reaction (3 hr, run simultaneously)</span></li></ol><ol class="c34"><li class="c14" value="1"><span class="c0">Use Phusion Polymerase</span></li><li class="c14"><span class="c0">Only 3 ul or so is needed for the PCR</span></li><li class="c14"><span class="c0">PCR volume can be 30 or 50 uL (50 works fine and gets us lots of DNA)</span></li></ol><ol class=""><li class="c3" value="5"><span class="c0">PCR purify (45 mins max)</span></li><li class="c3"><span class="c0">Check results on gel (40 mins max)</span></li></ol><ol class="c34"><li class="c14" value="1"><span class="c0">Gel extract if PCR results in multiple bands (1hr)</span></li></ol><ol class=""><li class="c3" value="7"><span class="c0">Digest the Aox-RFP construct (3 hours or overnight)</span></li><li class="c3"><span class="c0">&nbsp;</span></li></ol><p class="c1"><span class="c0 c2">Protocols</span></p><p class="c1"><span class="c0">Make sure all products are digested</span></p><ol class="c10"><li class="c3" value="1"><span class="c0">hyb.ompa is not digested, so start that today</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Ligation of AOX1a-FR2 to RFP-F2R (Scott)</span></p><p class="c1"><span class="c0">using AOX1a-FR2 from 9/27/2010 and RFP-F2R from 9/27/2010</span></p><p class="c1"><span class="c0 c12">Calculating equivalents:</span></p><p class="c1"><span class="c0">RFPF2R- [ 29 ng/uL]/678 bp= 0.0428 eq/uL</span></p><p class="c1"><span class="c0">AOX1a-FR2-[ 25 ng/uL]/1035 bp = 0.0242 eq/uL</span></p><p class="c1"><span class="c0">for linear ligations, use a 1:1 ratio of products</span></p><p class="c1"><span class="c0 c2">Ligation:</span></p><p class="c1"><span class="c0">1.1 uL of RFP F2R </span></p><p class="c1"><span class="c0">2 uL of AOX1a-FR2</span></p><p class="c1"><span class="c0">1 uL 10x Ligase Buffer</span></p><p class="c1"><span class="c0">5.4 uL H20</span></p><p class="c1"><span class="c0">0.5 uL T4 Ligase</span></p><p class="c1"><span class="c0 c7">Total=10 uL</span></p><p class="c1"><span class="c0">Leave RT for 1 hour</span></p><p class="c1"><span class="c0">Started 10:42 am</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Ligation of AOX1b-FR2 to RFP-F2R (Scott)</span></p><p class="c1"><span class="c0">using AOX1a-FR2 from 9/27/2010 and RFP-F2R from 9/27/2010</span></p><p class="c1"><span class="c0 c12">Calculating equivalents:</span></p><p class="c1"><span class="c0">RFPF2R- [ 29 ng/uL]/678 bp= 0.0428 eq/uL</span></p><p class="c1"><span class="c0">AOX1b-FR2-[ 21.3 ng/uL]/1047 bp = 0.0203 eq/uL</span></p><p class="c1"><span class="c0">for linear ligations, use a 1:1 ratio of products</span></p><p class="c1"><span class="c0 c2">Ligation:</span></p><p class="c1"><span class="c0">1 uL of RFP F2R</span></p><p class="c1"><span class="c0">2 uL of Aox1b-FR2</span></p><p class="c1"><span class="c0">1 uL 10x Ligase Buffer</span></p><p class="c1"><span class="c0">5.5 uL H20</span></p><p class="c1"><span class="c0">0.5 uL T4 Ligase</span></p><p class="c1"><span class="c0 c7">Total=10 uL</span></p><p class="c1"><span class="c0">Leave RT for 1 hour</span></p><p class="c1"><span class="c0">Started 10:42 am</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c2">Digest of HybB.Ompa (Christina)</span></p><p class="c1"><span class="c0">17 uL of Hyb.Ompa (58.2 ng/uL)</span></p><p class="c1"><span class="c0">8.5 uL h20</span></p><p class="c1"><span class="c0">3 uL 10x Buffer B (Promega)</span></p><p class="c1"><span class="c0">0.75 uL EcoRI</span></p><p class="c1"><span class="c0">0.75 uL XmaI</span></p><p class="c1"><span class="c0 c7">Total= 30 uL</span></p><p class="c1"><span class="c0">37c heating block (with water) for 3 hours</span></p><p class="c1"><span class="c0">Start: 10:00 pm</span></p><p class="c1"><span class="c0">End: 1pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0">Do the ligations once Hyb.Ompa is digested:</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c5">Ligation of AOX1a-FR to HybB.Ompa</span></p><p class="c1"><span class="c0 c5">using AOX1a-FR from 9/27/2010 and Hyb.Ompa from 9/29/2010</span></p><p class="c1"><span class="c0 c5 c12">Calculating equivalents:</span></p><p class="c1"><span class="c0 c5">HybB.Ompa - [ ng/uL]/474 bp= eq/uL</span></p><p class="c1"><span class="c0 c5">AOX1a-FR-[ 12 ng/uL]/1035 bp = &nbsp;0.0116 eq/uL</span></p><p class="c1"><span class="c0 c5">for linear ligations, use a 1:1 ratio of products</span></p><p class="c1"><span class="c0 c5">Ligation:</span></p><p class="c1"><span class="c0 c5"> uL of Aox1a-FR</span></p><p class="c1"><span class="c0 c5"> uL of Hyb.Ompa</span></p><p class="c1"><span class="c0 c5">1 uL 10x Ligase Buffer</span></p><p class="c1"><span class="c0 c5"> uL H20</span></p><p class="c1"><span class="c0 c5">0.5 uL T4 Ligase</span></p><p class="c1"><span class="c0 c5 c7">Total=10 uL</span></p><p class="c1"><span class="c0 c5">Leave RT for 1 hour</span></p><p class="c1"><span class="c0 c5">&nbsp;</span></p><p class="c1"><span class="c0 c5">Ligation of AOX1b-FR to HybB.Ompa</span></p><p class="c1"><span class="c0 c5">using AOX1b-FR from 9/17/2010 and HybB.Ompa from 9/29/2010</span></p><p class="c1"><span class="c0 c5 c12">Calculating equivalents:</span></p><p class="c1"><span class="c0 c5">HybB.Ompa- [ ng/uL]/474 bp= eq/uL</span></p><p class="c1"><span class="c0 c5">AOX1b-FR -[ 23 ng/uL]/1047bp = 0.022 eq/uL</span></p><p class="c1"><span class="c0 c5">for linear ligations, use a 1:1 ratio of products</span></p><p class="c1"><span class="c0 c5">Ligation:</span></p><p class="c1"><span class="c0 c5"> uL of Aox1b-FR </span></p><p class="c1"><span class="c0 c5"> uL of Hyb.Ompa</span></p><p class="c1"><span class="c0 c5">1 uL 10x Ligase Buffer</span></p><p class="c1"><span class="c0 c5"> uL H20</span></p><p class="c1"><span class="c0 c5">0.5 uL T4 Ligase</span></p><p class="c1"><span class="c0 c5 c7">Total=10 uL</span></p><p class="c1"><span class="c0 c5">Leave RT for 1 hour</span></p></body></html>
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+  </map>
+</a></p>
+<style type="text/css">ol{margin:0;padding:0}p{margin:0}.c3{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:72.0pt}.c7{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:36.0pt}.c1{color:#ffffff;font-size:10pt;font-family:Arial}.c0{line-height:1.15;text-indent:0pt;direction:ltr}.c6{list-style-type:disc}.c8{list-style-type:circle}.c4{text-decoration:underline}.c9{font-style:italic}.c5{list-style-type:lower-latin}.c11{background-color:#ffffff}.c10{text-decoration:line-through}.c2{font-weight:bold}</style></head><body class="c11"><p class="c0"><span class="c1 c2">9/13/2010</span></p><p class="c0"><span class="c1">Christina, Christian, Scott, Debika, Margo</span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1">Started from new aliquots of primers, 1:10 dilution of primer stock in MilliQ H2O. Changes are </span><span class="c1 c2">bolded</span><span class="c1">.</span></p><p class="c0"><span class="c1">Reactions: HybB F+R, OmpA F+R, AOX1A F&amp;R, and AOX1B F&amp;R.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4 c2">New PCR protocol:</span></p><p class="c0"><span class="c1">26.5 uL H2O</span></p><p class="c0"><span class="c1">10 uL </span><span class="c1 c2">TaQ 5X Reaction buffer</span></p><p class="c0"><span class="c1">5 uL forward primer </span></p><p class="c0"><span class="c1">5 uL reverse primer</span></p><p class="c0"><span class="c1">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c0"><span class="c1 c2">2 uL</span><span class="c1"> template DNA</span></p><p class="c0"><span class="c1">0.5 uL polymerase enzyme, </span><span class="c1 c2">TaQ</span></p><p class="c0"><span class="c1 c2">Total Volume= 50 uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">NOTE: We multiplied the entire protocol by 2 to get </span><span class="c1 c2">50 &nbsp;uL total volume</span><span class="c1"> for this attempt</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">We are reducing number of experiments at once (e.g. not all setups at once, just a few) - HybB, OmpA, AOX1A F&amp;R, and AOX1B F&amp;R.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">We are preparing two strips for PCR using this recipe and setup. We are then running them side-by-side in the PCR machine, one strip using the old cycle program, and one strip with two key modifications (suggested by Megan). We increased number of PCR cycles from 29 to 34 cycles, and reduced annealing temperature to 52 degrees. (If we go to low with the annealing temperature, we will be able to tell because we will see lots of bands on PCR.)</span></p><p class="c0"><span class="c1"></span><span class="c1 c4">Making gel for PCR</span></p><p class="c0"><span class="c1">1. Add 0.35g agarose to 35mL autoclaved water.</span></p><p class="c0"><span class="c1">2. Add 3.5mL 1X TBE</span></p><p class="c0"><span class="c1">3. &nbsp;Heat in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don&rsquo;t vaporize it, especially near your face!</span></p><p class="c0"><span class="c1">4. Add 38.5&#1405;L EtBr (edited from 45 uL, make 1000X)</span></p><p class="c0"><span class="c1">5. Pour gel and allow to harden. </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Goals: Purify the PCR reactions and look at them on a gel</span></p><p class="c0"><span class="c1 c2">&nbsp;</span></p><p class="c0"><span class="c1 c9 c2">See protocols page for PCR purification</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c2">note: unless we want to keep the DNA for future use (unless we NEED pure DNA), the purification step is not necessary. &nbsp;We could have just run the DNA without the purification step... EtBr is an intercalator that will only bind the DNA anyway.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4 c2">&nbsp;</span></p><p class="c0"><span class="c1 c2">(Pics from 9/11 and 9/13 were taped to the lab bench for group reference -- maybe get Megan and/or Richard to help us interpret.)</span></p><p class="c0"><span class="c1 c4 c2">&nbsp;</span></p><p class="c0"><span class="c1 c4">Starter cultures for cryostocks</span></p><p class="c0"><span class="c1">Made starter cultures (3uL CARB + 3mL LB + cells) from triple smear plates (9/10/2010), and put in the incubator for 24 hours.. &nbsp;Tomorrow (on 9/14/2010), make cryostocks from these starter cultures. Labelled according to insert.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c2">9/14/2010</span></p><p class="c0"><span class="c1">Results from 9/13/2010: &nbsp;The previous gel had fairly good AOX bands but after consulting with Richard we decided that the other samples weren&rsquo;t represented in the gel. Further, we should have seen primer bands near the end, so in future gels it&rsquo;s important not to let samples run off. </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Miniprep</span></p><p class="c0"><span class="c1">In order to prepare for another gel, a miniprep of the following samples from the starter cultures made on 9/13/2010 were run:</span></p><p class="c0"><span class="c1">1. PSB1A3</span></p><p class="c0"><span class="c1">2. AOX1a</span></p><p class="c0"><span class="c1">3. ompA</span></p><p class="c0"><span class="c1">4. hybB</span></p><p class="c0"><span class="c1">5. AOX1b</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">The contents were labeled and stored in the -20 freezer for further use in the gel. </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Crystocks</span></p><p class="c0"><span class="c1">Cryostocks were made from starter cultures grown on 9/13/2010. &nbsp;Two distinct colonies were taken from each plate - so there are duplicates of each. &nbsp;Stored in the -80C labelled: </span></p><p class="c0"><span class="c1">9-14 clg HybB (2)</span></p><p class="c0"><span class="c1">9-14 clg ompA (2)</span></p><p class="c0"><span class="c1">9-14 clg AOX1a (2)</span></p><p class="c0"><span class="c1">9-14 clg &nbsp;AOX1b (2)</span></p><p class="c0"><span class="c1">9-14 clg psb1A3 (2) [note: 1 of these starter cultures turned red, the other did not.]</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c2">9/15/2010</span></p><p class="c0"><span class="c1 c4">Goals:</span><span class="c1"> Perform PCR on the new minipreps from 9/14/2010 (using new 1:10 aliquots of primers from 9/13/2010)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">PCR Protocol</span></p><p class="c0"><span class="c1">26.5 uL H2O</span></p><p class="c0"><span class="c1">10 uL </span><span class="c1 c2">TaQ 5X Reaction buffer</span></p><p class="c0"><span class="c1">5 uL forward primer </span></p><p class="c0"><span class="c1">5 uL reverse primer</span></p><p class="c0"><span class="c1">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c0"><span class="c1 c2">2 uL</span><span class="c1"> template DNA</span></p><p class="c0"><span class="c1">0.5 uL polymerase enzyme, </span><span class="c1 c2">TaQ</span></p><p class="c0"><span class="c1 c2">Total Volume= 50 uL</span></p><p class="c0"><span class="c1">Notes: Wear gloves while doing the reaction. Keep all reagents on ice, including the PCR reactions. Add in the order of the protocol- get out the enzyme and place on ice right before you are about to use.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Reactions Done in PCR:</span></p><ol class="c6"><li class="c7" value="1"><span class="c1">HyBb F,R</span></li><li class="c7"><span class="c1">OmpA F,R</span></li><li class="c7"><span class="c1">Aox1a F,R</span></li><li class="c7"><span class="c1">Aox1a F,R2</span></li><li class="c7"><span class="c1">Aox1b F,R</span></li><li class="c7"><span class="c1">Aox1b F,R2</span></li></ol><p class="c0"><span class="c1">Notes: We are using a master mix of Water, TAQ Buffer, DNTPS, and TAQ. Add this to all the tubes (everything on ice), then add all the DNA reagents.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Results from nanospec:</span></p><p class="c0"><span class="c1 c4 c2">Sample&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Concentration (ng/uL)</span></p><p class="c0"><span class="c1">hybB&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;155.2 </span></p><p class="c0"><span class="c1">ompA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;70.2</span></p><p class="c0"><span class="c1">Aox1a&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;241.3</span></p><p class="c0"><span class="c1">Aox1b&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;253.2</span></p><p class="c0"><span class="c1">psb1A3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;53.6</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Making gel for PCR</span></p><p class="c0"><span class="c1">1. Add 0.35g agarose to 36 mL 1 x TBEautoclaved water.</span></p><p class="c0"><span class="c1">2. &nbsp;Heat in microwave until agarose dissolves. Allow to cool. Make sure there are NO VAPORS before adding EtBr. EtBr is an intercalator. Don&rsquo;t vaporize it, especially near your face!</span></p><p class="c0"><span class="c1">3. Add 35 &#1405;L EtBr (edited from 45 uL, make 1000X)</span></p><p class="c0"><span class="c1">4. Pour gel and allow to harden. </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c2">9/16/2010</span></p><p class="c0"><span class="c1 c4">Goals: </span></p><ol class="c6"><li class="c7" value="1"><span class="c1">Interpret gel results of the PCR from 9/15/2010</span></li><li class="c7"><span class="c1">Perform PCR (from 9/15/2010) again, this time with:</span></li></ol><ol class="c8"><li class="c3" value="1"><span class="c1">PHUSION Polymerase/Buffer</span></li><li class="c3"><span class="c1">RFP</span></li></ol><p class="c0"><span class="c1 c4">What we did:</span></p><p class="c0"><span class="c1">(Christina, Rob)</span></p><ol class="c6"><li class="c7" value="1"><span class="c1">Performed PCR with Phusion </span></li><li class="c7"><span class="c1">Reactions: </span></li></ol><ol class="c5"><li class="c3" value="1"><span class="c1">HyBb F,R</span></li><li class="c3"><span class="c1">OmpA F,R</span></li><li class="c3"><span class="c1">Aox1a F,R</span></li><li class="c3"><span class="c1">Aox1a F,R2</span></li><li class="c3"><span class="c1">Aox1b F,R</span></li><li class="c3"><span class="c1">Aox1b F,R2</span></li><li class="c3"><span class="c1">RFP F,R</span></li><li class="c3"><span class="c1">RFP F2,R</span></li></ol><ol class=""><li class="c7" value="7"><span class="c1">Performed miniprep of pSB1A3 from cell culture grown on 9/15/2010 (non-red)</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">PCR Protocol</span></p><p class="c0"><span class="c1">26.5 uL H2O</span></p><p class="c0"><span class="c1">10 uL </span><span class="c1 c2">PHUSION 5X Reaction buffer</span></p><p class="c0"><span class="c1">5 uL forward primer </span></p><p class="c0"><span class="c1">5 uL reverse primer</span></p><p class="c0"><span class="c1">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c0"><span class="c1 c2">2 uL</span><span class="c1"> template DNA</span></p><p class="c0"><span class="c1">0.5 uL polymerase enzyme, </span><span class="c1 c2">PHUSION</span></p><p class="c0"><span class="c1 c2">Total Volume= 50 uL</span></p><p class="c0"><span class="c1 c2">&nbsp;</span></p><p class="c0"><span class="c1 c4">Miniprep of pSB1A3</span></p><p class="c0"><span class="c1">1. Remove inoculation tubes from inoculation (37C shaker).</span></p><p class="c0"><span class="c1">2. Obtain P1 buffer from 4C refrigerator.</span></p><p class="c0"><span class="c1">3. Take centrifuge tubes and add 1.5mL of inoculated cells. </span></p><p class="c0"><span class="c1">4. Centrifuge at 3000 rpm (low) for 1-2 min. </span></p><p class="c0"><span class="c1">5. Spin until white pellet of cells forms at the bottom and liquid is more clear. </span></p><p class="c0"><span class="c1">6. Take off supernatant and discard. </span></p><p class="c0"><span class="c1">7. Repeat steps 4-6. </span></p><p class="c0"><span class="c1">8. Resuspend pelleted bacterial cells in 250&#1405;L P1 buffer. </span></p><p class="c0"><span class="c1">9. &nbsp;Add 250&#1405;L P2 buffer and invert 4-6 times (DO NOT VORTEX - doing so will shear DNA!)</span></p><p class="c0"><span class="c1">10. Add 350&#1405;L buffer N3 and immediately invert 4-6 times. </span></p><p class="c0"><span class="c1">11. Centrifuge for 10 min. at 13,000 rpm. </span></p><p class="c0"><span class="c1">12. Take supernatant and add to spin columns. </span></p><p class="c0"><span class="c1">13. Spin 30-60 sec. and discard flow through. </span></p><p class="c0"><span class="c1">14. Wash column with 750&#1405;L buffer PE and centrifuge 1 min. </span></p><p class="c0"><span class="c1">15. Discard flow through and centrifuge and additional minute.</span></p><p class="c0"><span class="c1">16. Please column into a clean 1.5mL microcentrifuge tube.</span></p><p class="c0"><span class="c1">17. Elute DNA by adding 30&#1405;L dH2O. </span></p><p class="c0"><span class="c1">18. Let stand for 1 min., then centrifuge for 1 min. </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Cryostock of hybB cells and pSB1A3 </span><span class="c1">(from cultures on 9/15/2010)</span></p><p class="c0"><span class="c1">900 uL of cells</span></p><p class="c0"><span class="c1">100 uL DMSO</span></p><p class="c0"><span class="c1 c2">Total 1 mL</span></p><p class="c0"><span class="c1">Mix, place in -80c Freezer</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">For next time</span></p><ol class="c6"><li class="c7" value="1"><span class="c1">Confirm all the predicted sizes match up with the observed sizes on the gel of the PCR from 9/15/2010 </span></li><li class="c7"><span class="c1">If all are confirmed (Christina has confirmed, Christian and Scott confirmed hybb, ompa), then proceed with PCR purifications of 40 uL of each reaction, then RE digests, then ligations to construct each construct</span></li></ol><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1 c2">9/17/2010</span></p><p class="c0"><span class="c1 c4">Goals:</span></p><p class="c0"><span class="c1">Since the PCR was run on 9/16 using Phusion (high fidelity), we need to:<br>A) Run gel (2 uL each)</span></p><p class="c0"><span class="c1">B) PCR purify (leftovers from the 50 uL stock= 48 uL)</span></p><p class="c0"><span class="c1">C) Nanospec</span></p><p class="c0"><span class="c1">D) Digest with restriction enzymes (runs overnight)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Protocols</span></p><p class="c0"><span class="c1 c4">&nbsp;</span></p><p class="c0"><span class="c1">Make a gel for running PCR products from 9/16/2010 to make sure the PCR was successful. </span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Making gel for PCR (1% agarose gels):</span></p><p class="c0"><span class="c1">Added 180 mL 1x TBE and 1.8g agarose</span></p><p class="c0"><span class="c1">Heated up until agarose was no longer visible</span></p><p class="c0"><span class="c1">Let cool (approx. 10 min) until there are NO MORE VAPORS</span></p><p class="c0"><span class="c1">Added 180 &#1405;L Ethidium bromide (1000X)</span></p><p class="c0"><span class="c1">Pour into gels and allow to harden (large wells hold about 35 mL).</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Note: it is ok to make 6 gels at once according to the recipe above and store them. It is not necessary to &ldquo;immediately&rdquo; use gels as long as they are stored properly. From now on, when you make 1% gels, just fill all 6 wells at once.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Gel of the PCR from 9/16/2010 was run today and imaged:</span></p><p class="c0"><span class="c1">Lane order: 100 bp ladder|aox1aFR|aox1aFR2|aox1bFR|apx1bFR2|HybB FR|ompa FR|RFP FR| RFP F2R|100 bp ladder</span></p><p class="c0"><span class="c1">volumes:</span></p><p class="c0"><span class="c1">loaded 4 ul of the aox reactions, 7 ul of ompa and hybB, and 5 ul of the RFP reactions</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c9 c2">See protocols page for PCR purification</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Nanospec results </span><span class="c1">(PCR from 9/16/2010)</span></p><p class="c0"><span class="c1">AOX1a-F,R = 99.3 ng/uL</span></p><p class="c0"><span class="c1">AOX1a-F,R2 = 50.0 ng/uL</span></p><p class="c0"><span class="c1">AOX1b-F,R = 48.5 ng/uL</span></p><p class="c0"><span class="c1">AOX1b-F,R2 = 42.1 ng/uL</span></p><p class="c0"><span class="c1">OmpA-F,R = 27.3 ng/uL</span></p><p class="c0"><span class="c1">RFP-F2,R = 10.8 ng/uL</span></p><p class="c0"><span class="c1">Hyb-F,R = 26.2 ng/uL</span></p><p class="c0"><span class="c1">RFP-F,R = 8.8 ng/uL</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Restriction Enzyme Digest General Process</span></p><p class="c0"><span class="c1">- check buffers (NEB site)</span></p><p class="c0"><span class="c1">-check if BSA required (NEB site)</span></p><p class="c0"><span class="c1">- Start w/ HybB and RFP (start @ step A) / pBS1A3 (start @ step D)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Recipe for : 50 uL RXN</span></p><p class="c0"><span class="c1">DNA (1-2 ug- based on nanospec results)</span></p><p class="c0"><span class="c1">1 uL each enzyme</span></p><p class="c0"><span class="c1">5 uL 10X buffer</span></p><p class="c0"><span class="c1">1 uL BSA(if needed)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Today we are digesting just the HybB-F,R and RFP-F,R in order to do the ligation on this simple construct. The rest of the PCR-ed, purified, and nanoscpec&rsquo;ed building blocks went into the freezer for later use.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Digest Recipe for Hyb</span><span class="c1">B</span></p><p class="c0"><span class="c1">40 uL HybB-F,R (based on the nanospec results above)</span></p><p class="c0"><span class="c1">1 uL EcoRI</span></p><p class="c0"><span class="c1">1 uL NotI</span></p><p class="c0"><span class="c1">4.7 uL 10X buffer for EcoRI [Edit 9/17/2010. Only EcoRIbuffer)</span></p><p class="c0"><span class="c1">1 uL 10x BSA (based on this specfic RE mix-- BSA is required)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Place into an eppe tube, pipet gently to mix. Incubate at 37 degrees in water bath overnight.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">RE Digest Recipe for RFP-F,R</span><span class="c1">r</span></p><p class="c0"><span class="c1">40 uL RFP-F,R (based on the nanospec results above) [Edit 9/17/2010, 110 ul not there, reduced to 40]</span></p><p class="c0"><span class="c1">1 uL SpeI (edited changed from notI to speI )</span></p><p class="c0"><span class="c1">1 uL NotI</span></p><p class="c0"><span class="c1">5 uL 10X buffer for EcoRI [Edit, only EcoRI buffer]</span></p><p class="c0"><span class="c1">1 uL10x BSA (based on this specfic RE mix-- BSA is required)</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Place into an eppendorf tube, pipette gently to mix. Incubate at 37 degrees in water bath overnight.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Results</span></p><p class="c0"><img height="366.0" src="https://static.igem.org/mediawiki/2010/8/82/9-12a.png" width="490.0"></p><p class="c0"><span class="c1 c9">Gel of multiple PCR reactions</span></p><p class="c0"><span class="c1 c9">100 bp Ladder| Aox1a FR| Aox1a FR2| Aox1b FR| Aox1b FR2| HybB FR| Ompa FR| RFP FR| RFP F2R| 100 bp ladder|</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">For Future</span></p><ol class="c6"><li class="c7" value="1"><span class="c1">We have a strategy for each construct, written on the board. We must digest each PCR product to build our building blocks, then construct each construct through a series of ligations, digest, PCRs etc (See 9.18.2010 for strategy)</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c2">9/18/2010</span></p><p class="c0"><span class="c1 c4">Goals</span></p><ol class="c6"><li class="c7" value="1"><span class="c1">PCR purify the hybB F,R a</span><span class="c1 c10">nd RFP F,R</span><span class="c1"> (rxns from 9/16/2010)</span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4">Notes:</span></p><ol class="c6"><li class="c7" value="1"><span class="c1">RFP F,R was accidently digested with NotI and not SpeI.</span></li><li class="c7"><span class="c1">What to do? We will need to add 1 uL of SpeI and run overnight, so that the SpeI site is cleaved. <br>Just in case we need this RFP, we PCR-purified it today and stored in freezer, clearly labeled PARTLY DIGESTED RFP. If we need to use this RFP, it will need to be digested by SpeI.</span></li><li class="c7"><span class="c1">RFP band on the gel pic from 9/17/2010 was faint and nanospec showed low conc (8.8 ng /uL) versus the higher yields of the other rxns. </span></li></ol><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1">Ran PCR purification on HybB, stored in freezer. This HybB was nanospec&rsquo;ed on Friday and had a concentration of &nbsp;26.2 ng/uL. This has been digested by the right RE&rsquo;s, EcoRI and NotI, and ready to be ligated with RFP when it has been appropriately digested.</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c9 c2">See protocols page for PCR purification</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c4 c2">BASIC PLAN FOR BUILDING PLASMID CONSTRUCTS </span></p><p class="c0"><span class="c1">from the primered building blocks, which are stored in our freezer</span></p><p class="c0"><span class="c1">A) RUN GEL - 2 uL each - to check size - this step completed for all building blocks</span></p><p class="c0"><span class="c1">B) PCR PURIFY leftovers - this step completed for all building blocks</span></p><p class="c0"><span class="c1">C) NANOSPEC - record concentrations - this step completed for all building blocks</span></p><p class="c0"><span class="c1 c2">D) DIGEST</span><span class="c1"> with restriction enzymes, overnight</span></p><p class="c0"><span class="c1 c2">E) PCR PURIFY</span><span class="c1"> digestion products</span></p><p class="c0"><span class="c1 c2">F) LIGATION</span><span class="c1"> of gene building blocks - we are starting with HybB &amp; RFP</span></p><p class="c0"><span class="c1 c2">G) PCR </span><span class="c1">ligation products, HybB-RFP</span></p><p class="c0"><span class="c1 c2">H) DIGEST</span><span class="c1"> products, HybB-RFP</span></p><p class="c0"><span class="c1 c2">I) RUN GEL</span><span class="c1"> on small amount of digestion products, HybB-RFP</span></p><p class="c0"><span class="c1 c2">J) PCR PURIFY</span><span class="c1"> the rest of the digestion products, HybB-RFP</span></p><p class="c0"><span class="c1 c2">K) DIGEST</span><span class="c1"> the vector - we are using pBS1A3</span></p><p class="c0"><span class="c1 c2">L) PCR PURIFY</span><span class="c1"> the vector</span></p><p class="c0"><span class="c1 c2">M) LIGATION </span><span class="c1">of gene with vector, HybB-RFP and pBS1A3</span></p><p class="c0"><span class="c1 c2">N) TRANSFORMATION </span><span class="c1">of plasmid into E. coli, run overnight</span></p><p class="c0"><span class="c1 c4">Note- </span><span class="c1"> Do a nanospec after PCR purifications</span></p><p class="c0"><span class="c1">&nbsp;</span></p><p class="c0"><span class="c1 c2">NOTES: </span></p><ol class="c6"><li class="c7" value="1"><span class="c1">Steps in BOLD are going to be repeated with each plasmid construct. Steps A,B, and C have already been done for each building block and won&rsquo;t be repeated unless there is a specific issue with a particular building block.</span></li><li class="c7"><span class="c1">Steps K and L do not necessarily need to wait until step J is completed to be run. They are listed this way so there is no confusion about what is being digested or PCR purified at each step. </span></li></ol>