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 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:

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:

SampleConcentration (ng/uL)

hybB155.2

ompA70.2

Aox1a241.3

Aox1b253.2

psb1A353.6

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

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)

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

PCR Purification