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Team:Gaston Day/Notebook
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Contents |
Procedures
A. Plasmid Prep
B. Digest
C. Ligation
D. Transformation
E. Gel Electrophoresis
A. 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.
B.== ==Digest
C.== ==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.
D.== ==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.
