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Revision as of 02:19, 22 October 2010

Procedures

Plasmid Prep

SOLUTIONS: TENS (make fresh each time) To 4.5 ml TE add: 250 ul 10% SDS 250 ul 2N NaOH Sodium Acetate 3.0M; pH 5.5 TE with 10 ug/ml of RNAse A

PROCEDURE: 1. Spin 1.5 ml of overnight culture for 30 sec in microfuge

2. Aspirate off all but 100 ul of the supernatant and resuspend the pellet by vortexting

3. Add 300 ul of TENS and mix by inversion. The solution should become viscous.

4. Add 150 ul of sodium acetate and votex. A fine white precipitate should form.

5. Centrifuge for 2.5 minutes at 10K.

6. TRANSFER the supernatant to a clean tube and add 2 volumes (1 ml) of room temperature EtOH.

7. Vortex and pellet DNA by centrifugation for 2-5 minutes at 10K.

8. Wash pellet with 70% ethanol and allow the pellet to dry.

9. Resuspend the pellet in TE with RNAseA.

10. Digest 5-10 ul as usual.

Digest

Ligation

1. Remove the 10X T4 DNA Ligase Reaction Buffer* from the freezer to thaw. You can remove the T4 DNA Ligase enzyme from the freezer at this point but leave the ligase in a cold box to keep it close to -20○ C. Thawing is fast if the buffer tube is immersed in room temperature water. Once thawed, agitate the 10X T4 DNA Ligase Reaction Buffer until all precipitate goes into solution.

2. Add 11ul of H2O to a 200ul PCR tube.

3. Add 2ul from each of the digest to the tube.**

4. Add 2ul of 10X T4 DNA Ligase Reaction Buffer to the tube.

5. Add 1ul of the T4 DNA Ligase to the tube.

6. The total volume in each tube should now be 20ul. Ensue the ligation is well mixed by flicking the tube. You can spin the tube in a microcentrifuge for a few seconds to collect the liquid in the bottom of the tube again.

*Repeated freeze-thaw cycles of the buffer can degrade the ATP in the buffer thereby making the ligation reaction less efficient. It is wise to aliquot the buffer into 10ul aliquots prior to freezing

**There is no need to purify the restriction digests via gel electrophoresis in any other method.

Transformation

1. Thaw tube of NEB 10-beta Competent E. coli cells on ice for 10 minutes.

2. Add 1-5ul containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. DO NOT VORTEX.

3. Place the mixture on ice for 30 minutes. Do not mix.

4. Heat shock at exactly 42○ C for exactly 30 seconds. Do not mix.

5. Place on ice for 5 minutes. Do not mix.

6. Pipette 950ul of room temperature SOC into the mixture.

7. Place at 37○ C for 60 minutes. Shave vigorously (250 rpm) or rotate.

8. Warm selection plates to 37○ C.

9. Mix the cells thoroughly by flicking the tube and inverting then perform several 10-fol serial dilutions in SOC.

10. Spread 50-100ul of each dilution onto a selection plate and incubate overnight at 37○ C for 24-36 hours of 25○ C for 48 hours.

March

March 2

Start cultures of:

I 765000 (Fe promoter, amp broth) LARGE FLASKS

E 1010 (RFP gene only, Kan broth) LARGE FLASKS

J 04450, amp SMALL

J 04450 kan SMALL

-Add .5 ml amp and kan to 50 ml bottle LB broth

March 3

All grew well

March 5 Make fresh TENS from plasmid preps

Miniprep Fe promoter + RFP

Teaching plasmid preps (Fe Prom, RFP gene, cRFP-A, cRFP-K) to Augustine E., Taylor G., Spencer J., Joey S., and Al Hall.

April

April 1

Restriction digest of Fe promoter (pSEX), cRFP, RFP gene

10x plasmid

2x 10x buffer 2(NEB)

1x Xba

1x Spe

6x dH20

20x

O/N 37o

April 2

5x loading buffer to each

Gel:

1 ladder – bands present

2 cRFP

3 Fe – bands present

4 RFP gene – bands present

Made TENS

Plated competent cells on plain LB

Plated pNICE on plain LB

Plasmid prep

April 3

Digest w/ spe/xba

5x DNA

1x Xba

1x Spe

2x dH2o

1x buffer 2

10x

April 5

Plasmid Preps of Fe, cRFP, and old Fe

Prepare cometent cells

Spin 5ml cells 30”

Drain sup

Resusp. In 300x CaCl2

Add 1x, 2x, 3x, 4x 50ng/ml plasmid pNICE

Plated on LB o/n

April 7

Selected colonies to grow on Kan. Grow o/n

April 14

Digest w/ Xba, pNICE, new Fe, old Fe, cRFP

Mix 37o @ 10

DNA 10x

Buffer 2x

Xba 1x

dH20 7x

20x

May

May 13

Start cRFP (K) growing

Start Fe pSEX

Cut w/ Xba, Spe, Xba/Spe

FepSEX

DNA 10x 10x 10x

Buffer 2x 2x 2x

Xba 1x Spe 1x Spe 1x

dH20 7x dH20 7x Xba 1x

H20 6x

20x 20x 20x

May 14

Ran 1% gel

1 ladder – 1KG

2 Spe

3 Xba

4 S/x ~1KG band present

Picture on digital camera

Repeat digest w/ new Fe