Site Directed Mutagenesis

From FreeBio

{The following protocol is based on Strategens' QuikChange Site-Directed Mutagenesis Kit, and can be used to introduce single-bp mutations. The kit is in the -20° C freezer; the magical XL1-Blue Supercompetent Cells and pUC18 Ctrl Plasmid are in the -80° freezer. The manufacturer's manual is available at here.}

I. Synthesis rxns

1. Set up synthesis rxns (total 50µl) in 0.5ml PCR tubes; make master mixes as appropriate:
   • 5µl 10x rxn buffer (kit)
   • 1µl 1:10 dilution dsDNA template (5-50 ng; dilution is for typical miniprep)
   • 1.25µl 10µM primer 1 (125 ng)
   • 1.25µl 10µM primer 2 (125 ng)
   • 1µl dNTP mix (kit)
   • 40.5µl ddH₂O
2. For control rxn (total 50µl):
   • 5µl 10x rxn buffer (kit)
   • 2µl pWhitescript 4.5-kb ctrl template (kit)
   • 1.25µl oligo ctrl primer #1
   • 1.25µl oligo ctrl primer #2
   • 1µl dNTP mix
   • 39.5µl ddH₂O
3. To each rxn, add 1µl PfuTurbo DNA polymerase (kit)
4. Program thermo-cycler according to the following guidelines; make sure to select hot-top/heated-lid on cycler:
   1. 30sec @ 95°C
   2. 30sec @ 95°C
   3. 1min @ 55°C
   4. 1min/kb template @ 68°C
   5. Return to step 2 for 11x (pt mutation);14x (single-aa change);17x (del or insert)
   6. 10°C for ever

II. Dpn 1 Digest

1. To each rxn, add 1µl DpnI restriction enzyme (kit); gently mix.
2. Transfer rxn from 0.5ml PCR tubes to 1.5ml Eppendorf tubes; spin down in microfuge for 1min @ 14k rpm
   {IMPORTANT: Do not try to centrifuge using 0.5ml PCR tubes, as the caps fall off, the tubes shatter, and the rxn solns are jettisoned out!}
3. Immediately incubate @ 37°C for 1h.
4. Meanwhile, make sure that the 42°C water-bath or heating-block is turned on.

III. Transformation

1. Gently thaw the magical XL1-Blue supercompetent cells on ice.
2. Aliquot 50µl of cells to pre-chilled 1.5ml Eppendorf tubes.
3. Add 1µl of Dpn-1 digested DNA to the cells. Swirl gently and incubate on ice for 30min.
4. Heat pulse the transformations for 45sec @ 42°C.
5. Immediately place on ice and incubate for 2'.
6. Add 0.5ml LB or SOC medium and incubate for 1h @ 37°C in the shaking incubator.
7. Plate 250µl on each of two plates for mutagenesis rxns;
   250µl on one plate for pWhitescript mutagenesis ctrl.
   {For the ctrl, plate on LB-amp plates w. 80µg/ml X-gal and 30mM IPTG.}
8. Incubate overnight @ 37°C

Posted dna1112 22:58, 20 Jun 2005 (EDT)