Mutagenesis - Plasmid DNA with Hydroxylamine
Jeff Lawrence (X06)
0.5M KnPO4, pH 6.0 10 mL 0.5 M K2HPO4 (approximate)
80 mL 0.5 M KH2PO4
5 mM EDTA 10 mL 0.5 M EDTA, pH 8.0
0.56 mL 4N NaOH
4.4 mL water (to 5 mL)
1. In small plastic Falcon tubes, mix 0.4 mL of Solution A, 0.8 mL of Solution B, and 0.8 mL of ddH2O. Prepare two tubes for each plasmid.
2. Add 2 µg of plasmid DNA to be mutagenized into each of the above tubes, and incubate one pair for 60 min, and the other for 90 min, at 70°.
3. Transfer the solutions to dialysis tubing; dialyze vs. 2 L of TE at 4° for 2-3 hr. Change the buffer and dialyze for another 2-3 hr. NOTE: It is critical to recovery of the plasmid DNA to do this dialysis before ethanol precipitation.
4. After dialysis, transfer each solution into a large (12 mL) plastic Falcon tube. Butanol extract until a final volume of 400 µL is reached.
5. Transfer the 400 µL of plasmid containing solution to 1.5 mL Eppendorf tubes Add 40 µL 3M NaOAc, pH 3.0 and 1.2 mL 95% ethanol. Freeze on dry ice, and rinse in 70% ethanol.
6. Resuspend the DNA pellet from each tube in 50 µL of sterile ddH2O, and use 5 µL of this to retransform your host of choice. Expect 4,000-5,000 colonies per plate from 10% of untreated controls, and 10-50% of this number for the 60 min and 90 min treated samples respectively.
Last Update: Thursday, 19-Jun-2014 11:54:47 PDT
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