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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-<style type="text/css">ol{margin:0;padding:0}p{margin:0}.c7{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:36.0pt}.c6{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:72.0pt}.c14{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:144.0pt}.c2{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:108.0pt}.c1{line-height:1.15;text-indent:0pt;direction:ltr}.c11{color:#000000;font-size:12pt;font-family:Arial}.c0{color:#000000;font-size:11pt;font-family:Arial}.c9{font-style:italic}.c12{text-align:center}.c15{background-color:#ffffff}.c16{list-style-type:circle}.c13{list-style-type:decimal}.c18{list-style-type:lower-roman}.c3{background-color:#00ff00}.c8{text-decoration:underline}.c5{list-style-type:lower-latin}.c19{background-color:#ffff00}.c17{background-color:#ff9900}.c20{background-color:#3d85c6}.c21{background-color:#00ffff}.c10{list-style-type:disc}.c4{font-weight:bold}</style></head><body class="c15"><p class="c1 c12"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></p><p class="c1 c12"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c8 c4">9/13/2010</span></p><p class="c1"><span class="c0 c4">Christina, Christian, Scott, Debika, Margo</span></p><p class="c1"><span class="c0 c4">&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 c4">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 c8 c4">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 c4">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 c4">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 c4">TaQ</span></p><p class="c1"><span class="c0 c4">Total Volume= 50 uL</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0">NOTE: We multiplied the entire protocol by 2 to get </span><span class="c0 c4">50 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">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">&nbsp;</span></p><p class="c1"><span class="c0 c8">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. 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 c8">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 &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 c4">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.</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c8">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">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 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 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 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">&nbsp;</span></p><p class="c1"><span class="c0 c4">(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 c4">&nbsp;</span></p><p class="c1"><span class="c0 c8 c4">Starter cultures for cryostocks</span></p><p class="c1"><span class="c0 c8 c4">&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.. 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 c8 c4">9/14/2010</span></p><p class="c1"><span class="c0">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&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 c8 c4">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 c8 c4">Crystocks</span></p><p class="c1"><span class="c0">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:</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 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 c4">9.15.2010</span></p><p class="c1"><span class="c0 c8">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 c8">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 c4">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 c4">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 c4">TaQ</span></p><p class="c1"><span class="c0 c4">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="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HyBb &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;OmpA &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aox1a &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aox1a &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R2</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aox1b &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aox1b &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 c8 c4">Results from nanospec:</span></p><p class="c1"><span class="c0 c8 c4">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 c8">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. 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 c4">9/16/2010</span></p><p class="c1"><span class="c0 c8">Goals:</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Interpret &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;gel results of the PCR from 9/15/2010</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Perform &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR (from 9/15/2010) again, this time with:</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c16"><li class="c6" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PHUSION &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Polymerase/Buffer</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;RFP</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><p class="c1"><span class="c0 c8">What we did:</span></p><p class="c1"><span class="c0">(Christina, Rob)</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Performed &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR with Phusion &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Reactions: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c5"><li class="c6" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HyBb &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;OmpA &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aox1a &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aox1a &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R2</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aox1b &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aox1b &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R2</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;RFP &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;RFP &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F2,R</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Performed &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 c8">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 c4">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 c4">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 c4">PHUSION</span></p><p class="c1"><span class="c0 c4">Total Volume= 50 uL</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c8">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. 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 c8">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 c4">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 c8">For next time</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Confirm &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;all the predicted sizes match up with the observed sizes on the gel &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;of the PCR from 9/15/2010 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;If &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;all are confirmed (Christina has confirmed, Christian and Scott &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;confirmed hybb, ompa), then proceed with PCR purifications of 40 uL &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;of each reaction, then RE digests, then ligations to construct each &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;construct</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">SEPTEMBER 17, 2010</span></p><p class="c1"><span class="c0 c8">Goals:</span></p><p class="c1"><span class="c0">Since the PCR was run on 9/16 using Phusion (high fidelity), we need to:</span></p><p class="c1"><span class="c0">A) Run gel (2 uL each)</span></p><p class="c1"><span class="c0">dB) 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 c8">Protocols</span></p><p class="c1"><span class="c0 c8">&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 c8">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 c8">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 c8">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 &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 c8">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 c8">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 c8">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 c8">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 c8">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 c8">Results</span></p><p class="c1"><span class="c0 c9">Gel of multiple PCR reactions</span></p><p class="c1"><span class="c0 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="c1"><span class="c0 c9">&nbsp;</span></p><p class="c1"><span class="c0 c8">For Future</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;have a strategy for each construct, written on the board. We must &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;digest each PCR product to build our building blocks, then construct &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;each construct through a series of ligations, digest, PCRs etc (See &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;9.18.2010 for strategy)</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">9.18.2010</span></p><p class="c1"><span class="c0 c8">Goals</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;purify the hybB F,R and &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;RFP F,R &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(rxns from 9/16/2010)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligate &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 c8">Notes:</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;RFP &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;F,R was accidently digested with NotI and not SpeI.</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;What &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;to do? We will need to add 1 uL of SpeI and run overnight, so that &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the SpeI site is cleaved.</span></li><li class="c7"><span class="c0">Just in case we need this RFP, we &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR-purified it today and stored in freezer, clearly labeled PARTLY &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DIGESTED RFP. If we need to use this RFP, it will need to be &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;digested by SpeI.</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;RFP &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;band on the gel pic from 9/17/2010 was faint and nanospec showed low &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;conc (8.8 ng /uL) versus the higher yields of the other rxns. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</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 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 c8">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 &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 c8 c4">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 c4">D) DIGEST</span><span class="c0"> with restriction enzymes, overnight</span></p><p class="c1"><span class="c0 c4">E) PCR PURIFY</span><span class="c0"> digestion products</span></p><p class="c1"><span class="c0 c4">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 c4">G) PCR </span><span class="c0">ligation products, HybB-RFP</span></p><p class="c1"><span class="c0 c4">H) DIGEST</span><span class="c0"> products, HybB-RFP</span></p><p class="c1"><span class="c0 c4">I) RUN GEL</span><span class="c0"> on small amount of digestion products, HybB-RFP</span></p><p class="c1"><span class="c0 c4">J) PCR PURIFY</span><span class="c0"> the rest of the digestion products, HybB-RFP</span></p><p class="c1"><span class="c0 c4">K) DIGEST</span><span class="c0"> the vector - we are using pBS1A3</span></p><p class="c1"><span class="c0 c4">L) PCR PURIFY</span><span class="c0"> the vector</span></p><p class="c1"><span class="c0 c4">M) LIGATION </span><span class="c0">of gene with vector, HybB-RFP and pBS1A3</span></p><p class="c1"><span class="c0 c4">N) TRANSFORMATION </span><span class="c0">of plasmid into E. coli, run overnight</span></p><p class="c1"><span class="c0 c8">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="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Steps &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;in BOLD are going to be repeated with each plasmid construct. Steps &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;A,B, and C have already been done for each building block and won&rsquo;t &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;be repeated unless there is a specific issue with a particular &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;building block.</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Steps &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;K and L do not necessarily need to wait until step J is completed to &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;be run. They are listed this way so there is no confusion about what &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;is being digested or PCR purified at each step. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</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="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mon: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Redo PCR RFP; digest RFP (O/N)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Tues: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;ligate hyB+RFP (O/N), Digest pSB1A3 (O/N)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Wed: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR ; digest (1hr); PCR purify (digests of hyb+rfp and pSB1a3) at &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;same time we run gel to check results; ligate O/N</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Thurs: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Tranformation of cells by hyb+RFP+psb1a3</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fri: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;check plates</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">9/20/2010</span></p><p class="c1"><span class="c0 c8">Goals</span><span class="c0">:</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c21">Redo &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR of mRFP F,R; mRFP F2,R</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c17">Digest &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;mRFP F,R; mRFP F2, R O/N (not possible until we get enzymes) </span><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c3">RUN &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GEL - 2 uL each - to check size - this step completed for all &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;building blocks</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c3">PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PURIFY leftovers - this step completed for all building blocks</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c3">NANOSPEC &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;- record concentrations - this step completed for all building &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;blocks</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c3">Transform &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;novablue with mRFP that ryan gave us (labeled mRFP H) to create our &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;own crytostock</span></li></ol><p class="c1"><span class="c0 c8">Notes:</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Ryan &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;gave us a fresh stock of mRFP plasmid</span><span class="c0">. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c8">We &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;only get this one</span><span class="c0">! &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;It is precious like gold or diamonds!</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Since &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the original mRFP has a Nde I site in the MCS, we are worried other &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;products may run at the same size. To avoid this worry, we will use &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;gel extraction to extract only our band of interest. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><p class="c1"><span class="c0 c8">mRFP</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;in &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the plasmid PET15B</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Use &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Novablue to create a cryostock. (transform, grow on plate, pick &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 c8">Protocols</span></p><p class="c1"><span class="c0 c8">&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 c8">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 c4">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 c4">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 c4">PHUSION</span></p><p class="c1"><span class="c0 c4">Total Volume= 50 uL</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c8">Ran PCR results (pre purification) on gel)</span></p><p class="c1"><span class="c0 c8">&nbsp;</span></p><p class="c1"><span class="c0 c8">&nbsp;</span></p><p class="c1"><span class="c0">12pm</span></p><p class="c1"><span class="c0 c8">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</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 c8">Results</span></p><ol class="c13"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Gel &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;picture of mRFP FR, F2R (Insert gel pic!)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c5"><li class="c6" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;bands of both PCRs (pre digest) were between approximately around &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;700-800 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><p class="c1"><span class="c0 c9">Gel of mRFP FR and mRFP F2R</span></p><p class="c1"><span class="c0 c9">100bp ladder|mRFP FR|mRFP F2R|Control</span></p><p class="c1"><span class="c0 c8">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 &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 c8">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 c8">For Future</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digest &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;first thing Tuesday!</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Richard &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;suggested getting two squirt bottles- 1 for water, 1 for bleach (to &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;kill cells)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Check &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;if transformation of Nova Blue with mRFP worked!</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">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 c8">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="c1"><span class="c0">3mL LB + 3uL 1000X CARB</span></p><p class="c1"><span class="c0">put in 37C incubator with shaking overnight</span></p><p class="c1"><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 c8">For 9/22/2010:</span></p><ol class="c16"><li class="c6" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Begin &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;digests 9am, do 3 hours insteadf of overnight, and ligation in &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;evening</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;create &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;cryostocks from starter cultures grown on 9/21/2010.</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Couldn&rsquo;t &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;start RE digest of RFP today- could not find RE&rsquo;s. Will ask Megan &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Wednesday morning.</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">9/22/2010- (Mitesh, Scott, Gita)</span></p><p class="c1"><span class="c0 c8">Goals</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digest &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;of RFP F,R</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;purify the digest</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligate &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 c8">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 c4">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 c8">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 &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 c8">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 c8">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 c9">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 c8">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 c4">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 c8">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 c4">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 c4">PHUSION</span></p><p class="c1"><span class="c0 c4">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 c8">For Future</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Take &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;out pcr of mrfp+hybB</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Run &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 uL on gel, along with just the RFP-FR and hybB-FR (1hr)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digest &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the construct, along with the vector psb1a3 at the same time (3 hrs)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligate &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the construct to psb1a3 (1 hr)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transform &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;into e coli (1.5 hrs)</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">9.23.2010 (Scott, Rob, Gita)</span></p><p class="c1"><span class="c0 c8">Goals</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Run &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR of RFP+hyBb on gel</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digest &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the construct, along with vecotr psb1a3</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;ligate &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the construct to psb1a3</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;transform &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 c8">Protocols</span></p><p class="c1"><span class="c0 c8">&nbsp;</span></p><p class="c1"><span class="c0 c8">Running PCR product on gel</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;pcr was left overnight at 12 c, and I am running 4 uL on a gel</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&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="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;I &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;could not see any stained DNA, even the ladder. This means the EtBr &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;degraded in the gels. Solution, suggested by Richard, is to keep a &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;bottle of 1% Agarose/TBE solution at RT. This solution represents &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the first step of making a gel. Then whenever we need a gel, we &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;start from the pre-made solution, add Etbr, and procede as normal. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Start with fresh gels.</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;products store in yellow box</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Building &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;blocks in blue rack</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c8">Making gel for PCR</span></p><p class="c1"><span class="c0">1. Heat 30 mL of 1% agarose solution 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 c8">Results</span></p><p class="c1"><span class="c0 c9">Gel from 9.23.2010.</span></p><p class="c1"><span class="c0 c9">100 bp ladder| RFP-FR +HybB PCR|</span></p><p class="c1"><span class="c0 c9">Predicted Size- 1070 bp</span></p><p class="c1"><span class="c0 c9">&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 c8">Making gel for PCR (Hammer Lab Apparatus)</span></p><p class="c1"><span class="c0">1. Heat 90 mL of 1% agarose solution 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 c8">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. 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 &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"><span class="c0 c9">Gel of RFP-FR + HybB FR PCR product (Before Gel Extraction)</span></p><p class="c1"><span class="c0 c9">30 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c1"><span class="c0 c9">Band to be excised is the 1100 bp band (2nd bright band from bottom)</span></p><p class="c1"><span class="c0 c9">&nbsp;</span></p><p class="c1"><span class="c0 c9">Gel of RFP-FR + HybB FR PCR product (After Gel Extraction)</span></p><p class="c1"><span class="c0 c9">30 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c1"><span class="c0 c9">&nbsp;</span></p><p class="c1"><span class="c0 c8">For Future-</span></p><p class="c1"><span class="c0 c9">Friday-</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;nanospec &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the gel extracted hyb+RFP construct (in blue box)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Run &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 uL on gel, see if 1100 bp is confirmed</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;if &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;so, continue with strategy. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">9/24/2010</span></p><p class="c1"><span class="c0 c8">Goals</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c20">nanospec &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the gel extracted hyb+RFP construct (in blue box)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c20">Run &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 uL on gel, see if 1100 bp is confirmed</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c3">if &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;so, digest the construct and vector psb1a3 (3 hrs); ligate (1hr), &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;transform (1.5 hours)</span></li></ol><p class="c1"><span class="c0 c3">&nbsp;</span></p><p class="c1"><span class="c0 c8">Protocols</span></p><p class="c1"><span class="c0 c8">&nbsp;</span></p><p class="c1"><span class="c0 c8">1 % Agarose Gel</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;I &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;found that the small gel trays need only 25-30 mL of solution (less &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;than 30)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;I &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;heated 30 mL of 1 % Agarose solution for approximately 45 seconds. I &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;then added 30 uL of EtBr (use blue gloves) and poured into the &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;prepared gel tray (tray inside of assembly, with combs). Cool for 1 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;hr. The gel will be done when there is a faint blue hue visible when &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;looking at it. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Gel &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Start: 9:10 am</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;End &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10:10 am</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c8">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 c9">Gel of Excised 1100 bp band of RFP-FR + HybB FR PCR product</span></p><p class="c1"><span class="c0 c9">4 uL of PCR product|9.5 uL of 100 bp ladder</span></p><p class="c1"><span class="c0 c9">Band to be excised is the 1100 bp band (2nd bright band from bottom)</span></p><p class="c1"><span class="c0 c9">&nbsp;</span></p><p class="c1"><span class="c0 c8">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 or 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 c4">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 c8">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 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 c4">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 c8">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 c4">30 uL of H20</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c8">Nanospec</span></p><p class="c1"><span class="c0">pSB1A3 Digest (purified) = 6.2</span><span class="c0 c19"> </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 c8">Ligation of pSB1A3 to [RFP-FR+ HybB-FR]</span><span class="c0"> (from 9.23.2010)</span></p><p class="c1"><span class="c0 c9">Calculating equivalents:</span></p><p class="c1"><span class="c0">pSB1A3- [ </span><span class="c0 c19">6.2</span><span class="c0"> ng/uL]/ 2100 bp= 3x10^-3 eq/uL</span></p><p class="c1"><span class="c0">hyBb+RFP-[ </span><span class="c0 c19">4.4</span><span class="c0"> ng/uL]/1100 bp = 4x10^-3 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="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;For &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;plasmid to linear ligations, use a 1:2 ratio of vector:products if &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the products are purified (1:4 if not)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c16"><li class="c6" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;E.g. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1 equivalent of vector to 2 equivalent of linear product</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c8">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 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 c4">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 c8">Transformation</span></p><p class="c1"><span class="c0 c8">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</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 c8">Notes for Future-</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aliquots &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;of BL21 and Novablue are in our -70c freezer</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Autoclave &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;fresh pipette tips- there is confusion over which tips fit what &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;pipettes- we need to calibrate to make sure our tips and pipettes &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 c4">SEPTEMBER 25, 2010</span></p><p class="c1"><span class="c0 c8">Goals-</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Begin &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;digesting the building blocks necessary to create the rest of the &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;constructs</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pick &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;colonies from the pSB1A3+Hyb+RFP transformation and grow overnight</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c8">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">&nbsp;</span></p><p class="c1"><span class="c0 c8">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 c4">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">&nbsp;</span></p><p class="c1"><span class="c0 c8">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 c4">Total=50 ul total</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">Went in at 2:55 pm</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c8">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 c4">Total=30 ul total</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">Went in at 3:05 pm</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c8">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 c4">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 c8">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 c4">Total=30 ul total</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">Went in at 3:10 pm</span></p><p class="c1"><span class="c0 c4">&nbsp;</span></p><p class="c1"><span class="c0 c8">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 c4">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 c19">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 c19">6,7 </span><span class="c0">pm</span></p><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c8">Preparing liquid culture of pSB1A3+hybB+RFP</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;mL of LB + 3 uL of Carb into a culture tube. Picked 2 colonies from &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;plates from 9.24.2010</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;O/N &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;at 37c in our incubator-shaker</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c8">For Future-</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;purify the digests</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;start &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;first round of ligations</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the ligation products</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c4">9/27/2010</span></p><p class="c1"><span class="c0 c8">Goals</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;purify digests of building blocks from 9/25/2010</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Start &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;first round of ligations for each construct</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Check &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;ligations on gel</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digest; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;troubleshoot ligations if necessary</span></li></ol><p class="c1"><span class="c0">&nbsp;</span></p><p class="c1"><span class="c0 c8">Protocols</span></p><p class="c1"><span class="c0 c8">&nbsp;</span></p><p class="c1"><span class="c0 c8">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 &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 c8">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 c8">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 c8">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 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 c9">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 c8">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 c8">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. 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 c8">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 c8">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 c4">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 c4">PHUSION</span></p><p class="c1"><span class="c0 c4">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 c8">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 c8">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 c4">9/28/2010</span></p><p class="c1"><span class="c0 c8">Goals</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;gel picture of the hybB+ompa is in the folder. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c16"><li class="c6" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;band is bright at 500 bp, and our predictions confirm the results. &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;purify the PCR (in the yellow box)</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Nanospec</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><p class="c1"><span class="c0 c8">Notes</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Christina+Rob &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR purified the hyb+ompa PCR.</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Richard &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;suggested we can PCR Aox+RFP, then pcr that to the hyb+ompa. This &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;wil take care of two constructs. The other two require Aox a/b to be &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;attached to the hybb+ompa construct</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;For &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the constructs that have Aox+RFP, we can do a ligation of Aox to &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;RFP; then, PCR that part and ligate it to the Hyb+ompa.</span></li></ol><ol class="c5"><li class="c6" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;For &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the AOX-RFP constructs:</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c18"><li class="c2" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligate &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aox1a-FR2 to RFP-F2R</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;LigateAox1b-FR2 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;to RFP-F2R</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;each ligation reaction</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;purify</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Check &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;results on gel</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class=""><li class="c14" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Gel &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;extract if PCR results in multiple bands</span></li><li class="c14"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c18"><li class="c2" value="11"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digest &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the Aox-RFP construct</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;purify</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligate &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the Aox-RFP digests to Hyb-Ompa digest</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the entire constructs</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class=""><li class="c6" value="3"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;For &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the HybB-Ompa-Aox constructs:</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c18"><li class="c2" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligate &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the HybB-Ompa to Aox1a-FR or Aox1b-FR</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the constructs</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Purify</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Check &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;results on gel</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c13"><li class="c14" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;if &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;multiple bands, gel extract</span></li><li class="c14"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c18"><li class="c2" value="9"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligate &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Hyb-Ompa-Aox to pSB1A3 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transform &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;into cells or PCR entire vector</span></li><li class="c2"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class=""><li class="c6" value="6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><p class="c1"><span class="c11 c4">Constructs</span></p><p class="c1"><span class="c4 c11">&nbsp;</span></p><p class="c1"><span class="c11 c4">&nbsp;</span></p><p class="c1"><span class="c11 c4">&nbsp;</span></p><p class="c1"><span class="c11 c4">&nbsp;</span></p><p class="c1"><span class="c11 c4">&nbsp;</span></p><p class="c1"><span class="c0 c4">9/29/2010</span></p><p class="c1"><span class="c0 c8">Goals</span></p><ol class="c13"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligate &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aox1a-FR2 to RFP-F2R (1hr)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;LigateAox1b-FR2 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;to RFP-F2R (1hr at same time as step 1)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digest &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HybB.Ompa (3 hours)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c5"><li class="c6" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligate &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the HybB-Ompa to Aox1a-FR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligate &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the HybB-Ompa to Aox1b-FR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c13"><li class="c7" value="7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;each ligation reaction (3 hr, run simultaneously)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c5"><li class="c6" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Use &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Phusion Polymerase</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Only &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3 ul or so is needed for the PCR</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;volume can be 30 or 50 uL (50 works fine and gets us lots of DNA)</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c13"><li class="c7" value="10"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;purify (45 mins max)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Check &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;results on gel (40 mins max)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c5"><li class="c6" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Gel &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;extract if PCR results in multiple bands (1hr)</span></li><li class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><ol class="c13"><li class="c7" value="15"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digest &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;the Aox-RFP construct (3 hours or overnight)</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li><li class="c7"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></li></ol><p class="c1"><span class="c0 c8">Protocols</span></p><p class="c1"><span class="c0">Make sure all products are digested</span></p><ol class="c10"><li class="c7" value="1"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;hyb.ompa &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 c8">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 c9">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 c8">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 c4">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 c8">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 c9">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 c8">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 c4">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 c8">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 c4">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 c8">Ligation of AOX1a-FR to HybB.Ompa</span></p><p class="c1"><span class="c0">using AOX1a-FR from 9/27/2010 and Hyb.Ompa from 9/29/2010</span></p><p class="c1"><span class="c0 c9">Calculating equivalents:</span></p><p class="c1"><span class="c0">HybB.Ompa - [ ng/uL]/474 bp= eq/uL</span></p><p class="c1"><span class="c0">AOX1a-FR-[ 12 ng/uL]/1035 bp = 0.0116 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 c8">Ligation:</span></p><p class="c1"><span class="c0">uL of Aox1a-FR</span></p><p class="c1"><span class="c0">uL of Hyb.Ompa</span></p><p class="c1"><span class="c0">1 uL 10x Ligase Buffer</span></p><p class="c1"><span class="c0">uL H20</span></p><p class="c1"><span class="c0">0.5 uL T4 Ligase</span></p><p class="c1"><span class="c0 c4">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 c8">Ligation of AOX1b-FR to HybB.Ompa</span></p><p class="c1"><span class="c0">using AOX1b-FR from 9/17/2010 and HybB.Ompa from 9/29/2010</span></p><p class="c1"><span class="c0 c9">Calculating equivalents:</span></p><p class="c1"><span class="c0">HybB.Ompa- [ ng/uL]/474 bp= eq/uL</span></p><p class="c1"><span class="c0">AOX1b-FR -[ 23 ng/uL]/1047bp = 0.022 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 c8">Ligation:</span></p><p class="c1"><span class="c0">uL of Aox1b-FR</span></p><p class="c1"><span class="c0">uL of Hyb.Ompa</span></p><p class="c1"><span class="c0">1 uL 10x Ligase Buffer</span></p><p class="c1"><span class="c0">uL H20</span></p><p class="c1"><span class="c0">0.5 uL T4 Ligase</span></p><p class="c1"><span class="c0 c4">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>
+<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>