Team:GeorgiaTech/WeekSeven

From 2010.igem.org

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 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.

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

PCR purifcation of the PCR reactions from 9/10/2010

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 30 µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

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.

PCR Results via Gel Electrophoresis

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!)

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 “old” PCR cycle program, and the H samples having been run with the “new” PCR cycle program (as described by today’s changes).

1. HybB with Forward and Reverse primers

2. AOX1a with Forward and Reverse primers

3. AOX1b with Forward and Reverse primers

4. OmpA with Forward and Reverse primers

The A lanes and its DNA ladder were more clearly visible than the H lanes. The visible results are:

1. Faint bands in A & H at ~ 6,000 bp

2. In A& H, very strong bands at 700 bp, strong bands at 3,000 bp, somewhat weak but still obvious band at 2,000 bp.

3. In A& H, very strong bands at 700 bp, strong bands at 3,000 bp, somewhat weak but still obvious band at 2,000 bp.

4. DNA may have “run off” 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.

(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:

1.         HyBb         F,R
2.         
3.         OmpA         F,R
4.         
5.         Aox1a         F,R
6.         
7.         Aox1a         F,R2
8.         
9.         Aox1b         F,R
10.         
11.         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:

1.         Interpret         gel results of the PCR from 9/15/2010
2.         
3.         Perform         PCR (from 9/15/2010) again, this time with:
4.         

1.                 PHUSION                 Polymerase/Buffer
2.                 
3.                 RFP
4.         

What we did:

(Christina, Rob)

1.         Performed         PCR with Phusion         
2.         
3.         Reactions:                  

1.                 HyBb                 F,R
2.                 
3.                 OmpA                 F,R
4.                 
5.                 Aox1a                 F,R
6.                 
7.                 Aox1a                 F,R2
8.                 
9.                 Aox1b                 F,R
10.                 
11.                 Aox1b                 F,R2
12.                 
13.                 RFP                 F,R
14.                 
15.                 RFP                 F2,R
16.         

1.         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

1.         Confirm         all the predicted sizes match up with the observed sizes on the gel         of the PCR from 9/15/2010         
2.         
3.         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

SEPTEMBER 17, 2010

Goals:

Since the PCR was run on 9/16 using Phusion (high fidelity), we need to:

A) Run gel (2 uL each)

dB) 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:

Lane order: 100 bp ladder|aox1aFR|aox1aFR2|aox1bFR|apx1bFR2|HybB FR|ompa FR|RFP FR| RFP F2R|100 bp ladder

volumes:

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

PCR Purification

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 30 µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

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

100 bp Ladder| Aox1a FR| Aox1a FR2| Aox1b FR| Aox1b FR2| HybB FR| Ompa FR| RFP FR| RFP F2R| 100 bp ladder|

For Future

1.         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

1.         PCR         purify the hybB F,R and         RFP F,R         (rxns from 9/16/2010)
2.         
3.         Ligate         hybB F,R and RFP F,R

Notes:

1.         RFP         F,R was accidently digested with NotI and not SpeI.
2.         
3.         What         to do? We will need to add 1 uL of SpeI and run overnight, so that         the SpeI site is cleaved.
4. 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.
5.         
6.         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.

PCR Purification

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 50 µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

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:

1.         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.
2.         
3.         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.         

Plan for this week:

Weekend: PCR purify hybB, run gel for pSB1A3

1.         Mon:         Redo PCR RFP; digest RFP (O/N)
2.         
3.         Tues:         ligate hyB+RFP (O/N), Digest pSB1A3 (O/N)
4.         
5.         Wed:         PCR ; digest (1hr); PCR purify (digests of hyb+rfp and pSB1a3) at         same time we run gel to check results; ligate O/N
6.         
7.         Thurs:         Tranformation of cells by hyb+RFP+psb1a3
8.         
9.         Fri:         check plates

9/20/2010

Goals:

1.         Redo         PCR of mRFP F,R; mRFP F2,R
2.         
3.         Digest         mRFP F,R; mRFP F2, R O/N (not possible until we get enzymes)         
4.         
5.         RUN         GEL - 2 uL each - to check size - this step completed for all         building blocks
6.         
7.         PCR         PURIFY leftovers - this step completed for all building blocks
8.         
9.         NANOSPEC         - record concentrations - this step completed for all building         blocks
10.         
11.         Transform         novablue with mRFP that ryan gave us (labeled mRFP H) to create our         own crytostock

Notes:

1.         Ryan         gave us a fresh stock of mRFP plasmid.         We         only get this one!         It is precious like gold or diamonds!
2.         
3.         Since         the original mRFP has a Nde I site in the MCS, we are worried other         products may run at the same size. To avoid this worry, we will use         gel extraction to extract only our band of interest.         

mRFP

1.         in         the plasmid PET15B
2.         
3.         Use         Novablue to create a cryostock. (transform, grow on plate, pick         colony, grow in liquid media, take cryostock)

Protocols

9am

PCR Protocol for mRFP F,R; mRFP F2, R

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

Ran PCR results (pre purification) on gel)

12pm

Heat shock transformation of the plasmids into our bacteria

10 սL Nova Blue cells + 5 սL of MRFP plasmid (from stock that Ryan gave us this morning).

1. Left cells and ligation reaction products on ice.

2. Added plasmid (5սL) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).

3. Left cells on ice for 30 min.

4. Applied heat shock of 45 seconds in 42C bath.

5. Put tubes on ice for 2 min.

6. Added 250 սL of LB (room temp.)

7. Incubated 1 hour at 37 C

8. Plated 100սL and left plate in the 37 degrees incubator

9. Incubated overnight at 37C.

Results

1.         Gel         picture of mRFP FR, F2R (Insert gel pic!)
2.         

1.                 The                 bands of both PCRs (pre digest) were between approximately around                 700-800                 
2.         

Gel of mRFP FR and mRFP F2R

100bp ladder|mRFP FR|mRFP F2R|Control

PCR Purification-mRFP

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 30 µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

Nanospec Results

mRFP, F: 137.2ng/uL

mRFP, F2: 132.3 ng/uL

For Future

1.         Digest         first thing Tuesday!
2.         
3.         Richard         suggested getting two squirt bottles- 1 for water, 1 for bleach (to         kill cells)
4.         
5.         Check         if transformation of Nova Blue with mRFP worked!

9/21/2010

Results from 9/20/2010

        Transformation of mRFP in NB cells successful (both plates).

Starter Cultures

Make starter cultures from both mRFP:NB plates, allow to grow overnight.

3mL LB + 3uL 1000X CARB

put in 37C incubator with shaking overnight

Received shipment of SpeI (200 uL) and XmaI (50 uL) from Promega, put in -20C freezer (Gaucher Lab).

For 9/22/2010:

1.                 Begin                 digests 9am, do 3 hours insteadf of overnight, and ligation in                 evening
2.                 
3.                 create                 cryostocks from starter cultures grown on 9/21/2010.
4.                 
5.                 Couldn’t                 start RE digest of RFP today- could not find RE’s. Will ask Megan                 Wednesday morning.
6.         

9/22/2010- (Mitesh, Scott, Gita)

Goals

1.         Digest         of RFP F,R
2.         
3.         PCR         purify the digest
4.         
5.         Ligate         hyBB FR, to RFP F,R

Notes: made a stock of 1 ug/ul BSA for easier RE digests

I am following the suggested RE protocol listed on the Promega literature that came with the RE’s

RE Digest Recipe for RFP-F,Rr

4.5 uL H20

2uL 10X Promega Buffer D

10 uL RFP-F,R ( from 9.20.2010, 137 ug/ul)

2 uL BSA (0ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI (edited changed from notI to speI )

0.75 uL NotI

Total=20 ul total

Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)

Start time is 10:35 am

End time should be 1:35 pm

Purification of RFP digest

1. Added 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 30 µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

Nanospec

RFP Digest (purified)- 41 ng/uL

Ligation of RFP-FR to HybB-FR

using hybB from 9/18/2010 and RFP from 9/21/2010

Calculating equivalents:

RFP- [41 ng/uL]/678 bp= 0.06 eq/uL

hyBb-[26 ng/uL]/393 bp = 0.06 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

2 uL of RFP FR (SpeI, NotI digested; purified)

2 uL of HybB FR (EcoRI, NotI digested; purified)

1 uL 10x Ligase Buffer

4.5 uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour

Start time: 3:20 pm

End time: 4:20 pm

PCR of hybB+Mrfp construct

3 uL of ligation reaction

25.5 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)

0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

Start: 5pm

End: Thursday

For Future

1.         Take         out pcr of mrfp+hybB
2.         
3.         Run         2 uL on gel, along with just the RFP-FR and hybB-FR (1hr)
4.         
5.         Digest         the construct, along with the vector psb1a3 at the same time (3 hrs)
6.         
7.         Ligate         the construct to psb1a3 (1 hr)
8.         
9.         Transform         into e coli (1.5 hrs)

9.23.2010 (Scott, Rob, Gita)

Goals

1.         Run         PCR of RFP+hyBb on gel
2.         
3.         Digest         the construct, along with vecotr psb1a3
4.         
5.         ligate         the construct to psb1a3
6.         
7.         transform         into e. coli

Notes:

Predicted size of hyb+MRFP~1000bp

Protocols

Running PCR product on gel

1.         The         pcr was left overnight at 12 c, and I am running 4 uL on a gel
2.         
3.         4          uL sample + 1 uL running dye

Notes:

1.         I         could not see any stained DNA, even the ladder. This means the EtBr         degraded in the gels. Solution, suggested by Richard, is to keep a         bottle of 1% Agarose/TBE solution at RT. This solution represents         the first step of making a gel. Then whenever we need a gel, we         start from the pre-made solution, add Etbr, and procede as normal.         Start with fresh gels.
2.         
3.         PCR         products store in yellow box
4.         
5.         Building         blocks in blue rack

Making gel for PCR

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’t vaporize it, especially near your face!

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

3. Pour gel and allow to harden.

Results

Gel from 9.23.2010.

100 bp ladder| RFP-FR +HybB PCR|

Predicted Size- 1070 bp

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.

Making gel for PCR (Hammer Lab Apparatus)

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’t vaporize it, especially near your face!

2. Add 90 սL EtBr (edited from 45 uL, make 1000X)

3. Pour gel and allow to harden.

(I did 100 mL, but found the actual volume to be around 80 to 90 mL)

Start: 4:10 pm

End: 5:10 pm

Gel Extraction Protocol

1. Excised DNA fragment from the agarose gel with a clean, sharp scalpel.

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 μL).

3. Incubated at 50ºC for 10 min (or until the gel slice had completely dissolved). To help dissolve gel, mixed by vortexing the tube every 2 – 3 min during the incubation.

4. After the gel slice has completely dissolved, checked that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).

5. Added 1 gel volume of isopropanol to the sample and mixed.

6. Placed a QIAquick spin column in a provided 2 mL collection tube.

7. To bind DNA, applied the sample to the QIAquick column, and centrifuged for 1 min.

8. Discarded flow-through and placed QIAquick column back in the same collection tube.

9. Recommended: Added 0.5 mL of Buffer GQ to QIAquick column and centrifuged for 1 min.

10. To wash, added 0.75 mL of Buffer PE to QIAquick column and centrifuged for 1 min.

11. Discarded the flow-through and centrifuged the QIAquick column for an additional 1 min at 17,900 x g (13,000 rpm).

12. Placeed QIAQuick column into a clean 1.5 mL microcentrifuge tube.

13. To elute DNA, added 30 μL water (pH 7.0 – 8.5), let the column stand for 1 min, and then centrifuged for 1 min.

Gel of RFP-FR + HybB FR PCR product (Before Gel Extraction)

30 uL of PCR product|9.5 uL of 100 bp ladder

Band to be excised is the 1100 bp band (2nd bright band from bottom)

Gel of RFP-FR + HybB FR PCR product (After Gel Extraction)

30 uL of PCR product|9.5 uL of 100 bp ladder

For Future-

Friday-

1.         nanospec         the gel extracted hyb+RFP construct (in blue box)
2.         
3.         Run         2 uL on gel, see if 1100 bp is confirmed
4.         
5.         if         so, continue with strategy.         

9/24/2010

Goals

1.         nanospec         the gel extracted hyb+RFP construct (in blue box)
2.         
3.         Run         2 uL on gel, see if 1100 bp is confirmed
4.         
5.         if         so, digest the construct and vector psb1a3 (3 hrs); ligate (1hr),         transform (1.5 hours)

Protocols

1 % Agarose Gel

1.         I         found that the small gel trays need only 25-30 mL of solution (less         than 30)
2.         
3.         I         heated 30 mL of 1 % Agarose solution for approximately 45 seconds. I         then added 30 uL of EtBr (use blue gloves) and poured into the         prepared gel tray (tray inside of assembly, with combs). Cool for 1         hr. The gel will be done when there is a faint blue hue visible when         looking at it.         
4.         
5.         Gel         Start: 9:10 am
6.         
7.         End         10:10 am

Nanospec of Gel Extract from RFP+HybB PCR

19.2 ng/uL

(inset gel pic from etbr lab)

Gel of Excised 1100 bp band of RFP-FR + HybB FR PCR product

4 uL of PCR product|9.5 uL of 100 bp ladder

Band to be excised is the 1100 bp band (2nd bright band from bottom)

RE Digest Recipe for HybB +RFP PCR (Mitesh did this today)

3.5 uL H20

3uL 10X Promega Buffer E or Multicore

20uL HybB+RFP (excised 1100 bp badn from PCR) ( from 9.23.2010, 19 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL EcoRI

Total=30 ul total

Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)

RE Digest Recipe for pSB1A3 (Debika did this today)

3.5 uL H20 (edit 9/24/2010- should be 8.5 uL)

5uL 10X Promega Buffer E or Multicore

10 uL pSB1A3 (9.16.2010, 100 ng/uL)

5 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL EcoRI

Total=50 ul total

Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)

PCR Purfiy the two digests (RFP+Hyb construct, pSB1A3)

Note- We have had the best results when eluting in 30 uL of H20

Nanospec

pSB1A3 Digest (purified) = 6.2 ng/uL

RFP+HYBB Digest (purified)= 4.4ng/uL

Ligation of pSB1A3 to [RFP-FR+ HybB-FR] (from 9.23.2010)

Calculating equivalents:

pSB1A3- [ 6.2 ng/uL]/ 2100 bp= 3x10^-3 eq/uL

hyBb+RFP-[ 4.4 ng/uL]/1100 bp = 4x10^-3 eq/uL

1.         For         plasmid to linear ligations, use a 1:2 ratio of vector:products if         the products are purified (1:4 if not)
2.         

1.                 E.g.                 1 equivalent of vector to 2 equivalent of linear product
2.         

Ligation Protocol:

3 uL of RFP + HYB (~ 2 to 3 uL)

2 uL of Plasmid pSB1A3 (~ 1/2x amount of linear product on eq/uL basis)

1 uL 10x Ligase Buffer

3.5 uL H20

0.5 uL T4 Ligase (enzyme-- keep in freezer-- add last of all)

Total=10 uL

Leave RT for 1 hour

Start: 5:23 pm

Transformation

Heat shock transformation of the plasmids into our bacteria

10 սL Nova Blue cells + 5 սL of Ligation Reaction

1. Left cells and ligation reaction products on ice.

2. Added plasmid (5սL) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).

3. Left cells on ice for 30 min.

4. Applied heat shock of 45 seconds in 42C bath.

5. Put tubes on ice for 2 min.

6. Added 250 սL of LB (room temp.)

7. Incubated 1 hour at 37 C

8. Plated 100սL (x2 plates) and left plates in the 37 degrees incubator

9. Incubated overnight at 37C.

Notes for Future-

1.         Aliquots         of BL21 and Novablue are in our -70c freezer
2.         
3.         Autoclave         fresh pipette tips- there is confusion over which tips fit what         pipettes- we need to calibrate to make sure our tips and pipettes         are giving us the volumes we want

SEPTEMBER 25, 2010

Goals-

1.         Begin         digesting the building blocks necessary to create the rest of the         constructs
2.         
3.         Pick         colonies from the pSB1A3+Hyb+RFP transformation and grow overnight

Nanospec results (From tubes dated 9/17/2010)

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

RE Double Digest Recipe for RFP-F2,R

0 uL H20 (plenty in the RFP-F2,R tube, which is very dilute)

5 uL 10X Promega Buffer D

40 uL RFP-F2,R ( from 9.17.2010, 10.8 ng/ul)

5 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL NdeI

Total=51.5 ul total

Start :4pm

End: 7 pm

RE Double Digest Recipe for OmpA F,R

1.5 uL H20

5 uL 10X Promega Multi-Core Buffer

37 uL OmpA-F,R ( from 9.17.2010, 27.3 ng/ul)

5 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL NotI

0.75 uL XmaI

Total=50 ul total

Went in at 2:55 pm

RE Double Digest Recipe for AOX1a F,R

12.5 uL H20

3 uL 10X Promega Buffer B

10 uL AOX1a-F,R ( from 9.17.2010, 99.3 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL XmaI

Total=30 ul total

Went in at 3:05 pm

RE Double Digest Recipe for AOX1a F,R2

2.5 uL H20

3 uL 10X Promega Multi Core Buffer

20 uL AOX1a-F,R2 ( from 9.17.2010, 50 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL NdeI

0.75 uL XmaI

Total=30 ul total

Start 4 pm

RE Double Digest Recipe for AOX1b F,R

2.5 uL H20

3 uL 10X Promega Buffer B

20 uL AOX1b-F,R ( from 9.17.2010, 48.5 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL XmaI

Total=30 ul total

Went in at 3:10 pm

RE Double Digest Recipe for AOX1b F,R2

0 uL H20

3 uL 10X Promega Multi Core Buffer

22.5 uL AOX1b-F,R ( from 9.17.2010, 42.1 ng/ul)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL NdeI

0.75 uL XmaI

Total=30 ul total

Start 4 pm

Run 3 hours in the heating blockin our lab (37c, put water in the heating blocks)

Start time is 3, 4pm (two sets of reactions, noted in the individual descriptions)

End time should be 6,7 pm

Preparing liquid culture of pSB1A3+hybB+RFP

1.         3         mL of LB + 3 uL of Carb into a culture tube. Picked 2 colonies from         plates from 9.24.2010
2.         
3.         O/N         at 37c in our incubator-shaker

For Future-

1.         PCR         purify the digests
2.         
3.         start         first round of ligations
4.         
5.         PCR         the ligation products

9/27/2010

Goals

1.         PCR         purify digests of building blocks from 9/25/2010
2.         
3.         Start         first round of ligations for each construct
4.         
5.         Check         ligations on gel
6.         
7.         Digest;         troubleshoot ligations if necessary

Protocols

PCR Purification of Digests from 9/25/2010 (Ompa FR , AOX1a/b FR and FR2, RFP F2R)

Reactions I am purifying:

RFP-F2R (NdeI, SpeI)

OmpA FR (NotI, XmaI)

Aox1a-FR (Xma, SpeI)

Aox1a-FR2 (Xma, NdeI)

Aox1b-FR (Xma, SpeI)

Aox1b-FR2 (Xma, NdeI)

1. Added 5 volumes of Buffer PBI to 1 volume of the sample and mix (in a clean 1.5 mL eppendorf)

2. Checked that the color of the mixture was yellow (similar to Buffer PBI without the PCR sample).

3.. To bind DNA, transferred the sample to the column and centrifuge at 17,900g for 30 – 60 secs.

4. Discarded flow-through. Placed the column back in the same tube.

5. To wash, added 0.75 mL Buffer PE to the column and centrifuged for 30 – 60 secs.

6. Discarded flow-through and place the column back in the same tube. Centrifuged the column for an additional 1 min.

7. Placed the column in a clean 1.5 mL microcentrifuge tube.

8. To elute DNA, 30 µL autoclaved milliQ water to the center of the membrane, let the column sit for 1 min, and then centrifuged.

Nanospec of Purified digests

RFP-F2R (NdeI, SpeI)= 28.6 ng/uL

OmpA FR (NotI, XmaI)= 15 ng/uL

Aox1a-FR (Xma, SpeI)= 12 ng/uL

Aox1a-FR2 (Xma, NdeI)= 25 ng/uL

Aox1b-FR (Xma, SpeI)= 23 ng/uL

Aox1b-FR2 (Xma, NdeI)= 21.3 ng/uL

Ligations

hyb= [26.2 ng/ul ]/ 393 bp = 0.067 eq/uL

ompa = [15 ng/uL]/81 bp = 0.185 eq/uL

Ligation of HyBb+ Ompa

5.7 uL H20

2 uL of HybB

0.8 uL of Ompa

1 uL 10x Ligase Buffer

0.5 uL T4 Ligase (enzyme-- keep in freezer-- add last of all)

Total 10 uL

RT for 1 hr. - Start time: 11:25 am

Aliquot T4 Buffer:

Make aliquots (5 uL) of the T4 Ligase Buffer so as to not continuously repeat freeze-thaw cycles.

USE FROM ALIQUOTS FROM NOW ON - NOT THE GREEN-CAPPED EPPENDORF.

Miniprep of pSB1A3+hybB+ompA

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.

Making a 1 % gel

25*4=100 mL

We found that we had extra left over.

Start at 1:13 pm

End: 2:13 pm

PCR of Hyb+OmpA ligation

3 uL of product of ligation reaction

25.5 uL H2O

10 uL PHUSION 5X Reaction buffer

5 uL HybB-F forward primer

5 uL OmpA-R reverse primer

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

0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

Start: 12:57 pm

End: 3:00 pm (approx)

Plan:

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).

Gel Picture

insert gel pic from computer

band around 500 bp.

Nanospec PCR

Christina

326 ng/uL

9/28/2010

Goals

1.         The         gel picture of the hybB+ompa is in the folder.         
2.         

1.                 The                 band is bright at 500 bp, and our predictions confirm the results.                 
2.                 
3.                 PCR                 purify the PCR (in the yellow box)
4.                 
5.                 Nanospec
6.         

Notes

1.         Christina+Rob         PCR purified the hyb+ompa PCR.
2.         
3.         Richard         suggested we can PCR Aox+RFP, then pcr that to the hyb+ompa. This         wil take care of two constructs. The other two require Aox a/b to be         attached to the hybb+ompa construct
4.         
5.         For         the constructs that have Aox+RFP, we can do a ligation of Aox to         RFP; then, PCR that part and ligate it to the Hyb+ompa.

1.                 For                 the AOX-RFP constructs:
2.                 

1.                         Ligate                         Aox1a-FR2 to RFP-F2R
2.                         
3.                         LigateAox1b-FR2                         to RFP-F2R
4.                         
5.                         PCR                         each ligation reaction
6.                         
7.                         PCR                         purify
8.                         
9.                         Check                         results on gel
10.                         

1.                                 Gel                                 extract if PCR results in multiple bands
2.                         

11.                         
12.                         Digest                         the Aox-RFP construct
13.                         
14.                         PCR                         purify
15.                         
16.                         Ligate                         the Aox-RFP digests to Hyb-Ompa digest
17.                         
18.                         PCR                         the entire constructs
19.                 

3.                 
4.                 For                 the HybB-Ompa-Aox constructs:
5.                 

1.                         Ligate                         the HybB-Ompa to Aox1a-FR or Aox1b-FR
2.                         
3.                         PCR                         the constructs
4.                         
5.                         PCR                         Purify
6.                         
7.                         Check                         results on gel
8.                         

1.                                 if                                 multiple bands, gel extract
2.                         

9.                         
10.                         Ligate                         Hyb-Ompa-Aox to pSB1A3                         
11.                         
12.                         Transform                         into cells or PCR entire vector
13.                 

Constructs

9/29/2010

Goals

1.         Ligate         Aox1a-FR2 to RFP-F2R (1hr)
2.         
3.         LigateAox1b-FR2         to RFP-F2R (1hr at same time as step 1)
4.         
5.         Digest         HybB.Ompa (3 hours)
6.         

1.                 Ligate                 the HybB-Ompa to Aox1a-FR                 
2.                 
3.                 Ligate                 the HybB-Ompa to Aox1b-FR                 
4.         

7.         
8.         PCR         each ligation reaction (3 hr, run simultaneously)
9.         

1.                 Use                 Phusion Polymerase
2.                 
3.                 Only                 3 ul or so is needed for the PCR
4.                 
5.                 PCR                 volume can be 30 or 50 uL (50 works fine and gets us lots of DNA)
6.         

10.         
11.         PCR         purify (45 mins max)
12.         
13.         Check         results on gel (40 mins max)
14.         

1.                 Gel                 extract if PCR results in multiple bands (1hr)
2.         

15.         
16.         Digest         the Aox-RFP construct (3 hours or overnight)
17.         
18.         

Protocols

Make sure all products are digested

1.         hyb.ompa         is not digested, so start that today

Ligation of AOX1a-FR2 to RFP-F2R (Scott)

using AOX1a-FR2 from 9/27/2010 and RFP-F2R from 9/27/2010

Calculating equivalents:

RFPF2R- [ 29 ng/uL]/678 bp= 0.0428 eq/uL

AOX1a-FR2-[ 25 ng/uL]/1035 bp = 0.0242 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

1.1 uL of RFP F2R

2 uL of AOX1a-FR2

1 uL 10x Ligase Buffer

5.4 uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour

Started 10:42 am

Ligation of AOX1b-FR2 to RFP-F2R (Scott)

using AOX1a-FR2 from 9/27/2010 and RFP-F2R from 9/27/2010

Calculating equivalents:

RFPF2R- [ 29 ng/uL]/678 bp= 0.0428 eq/uL

AOX1b-FR2-[ 21.3 ng/uL]/1047 bp = 0.0203 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

1 uL of RFP F2R

2 uL of Aox1b-FR2

1 uL 10x Ligase Buffer

5.5 uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour

Started 10:42 am

Digest of HybB.Ompa (Christina)

17 uL of Hyb.Ompa (58.2 ng/uL)

8.5 uL h20

3 uL 10x Buffer B (Promega)

0.75 uL EcoRI

0.75 uL XmaI

Total= 30 uL

37c heating block (with water) for 3 hours

Start: 10:00 pm

End: 1pm

Do the ligations once Hyb.Ompa is digested:

Ligation of AOX1a-FR to HybB.Ompa

using AOX1a-FR from 9/27/2010 and Hyb.Ompa from 9/29/2010

Calculating equivalents:

HybB.Ompa - [ ng/uL]/474 bp= eq/uL

AOX1a-FR-[ 12 ng/uL]/1035 bp = 0.0116 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

uL of Aox1a-FR

uL of Hyb.Ompa

1 uL 10x Ligase Buffer

uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour

Ligation of AOX1b-FR to HybB.Ompa

using AOX1b-FR from 9/17/2010 and HybB.Ompa from 9/29/2010

Calculating equivalents:

HybB.Ompa- [ ng/uL]/474 bp= eq/uL

AOX1b-FR -[ 23 ng/uL]/1047bp = 0.022 eq/uL

for linear ligations, use a 1:1 ratio of products

Ligation:

uL of Aox1b-FR

uL of Hyb.Ompa

1 uL 10x Ligase Buffer

uL H20

0.5 uL T4 Ligase

Total=10 uL

Leave RT for 1 hour