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 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:

1. HyBb F,R
2. OmpA F,R
3. Aox1a F,R
4. Aox1a F,R2
5. Aox1b F,R
6. 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. Perform PCR (from 9/15/2010) again, this time with:

1. PHUSION Polymerase/Buffer
2. RFP

What we did:

(Christina, Rob)

1. Performed PCR with Phusion
2. Reactions:

1. HyBb F,R
2. OmpA F,R
3. Aox1a F,R
4. Aox1a F,R2
5. Aox1b F,R
6. Aox1b F,R2
7. RFP F,R
8. RFP F2,R

7. 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. 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:

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

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

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)

Notes:

1. RFP F,R was accidently digested with NotI and not SpeI.
2. 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.
3. 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:

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. 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.