Editing Quick Change (section)

== PCR  ==

1. Synthesize two complimentary oligonucleotides containing the desired mutation, flanked by unmodified nucleotide sequence. Purify these oligonucleotide "primers" prior to use in the following steps. 

2. Prepare the control reaction as indicated below: 

5 ÎŒl of 10Ã reaction buffer (see Preparation of Media and Reagents)<br>2 ÎŒl (10 ng) of pWhitescript 4.5-kb control plasmid (5 ng/ÎŒl)<br>1.25 ÎŒl (125 ng) of oligonucleotide control primer #1 

[34-mer (100 ng/ÎŒl)] 1.25 ÎŒl (125 ng) of oligonucleotide control primer #2<br>[34-mer (100 ng/ÎŒl)] 1 ÎŒl of dNTP mix<br>39.5 ÎŒl of double-distilled water (ddH2O) to a final volume of 50 ÎŒl 

Then add<br>1 ÎŒl of PfuTurbo DNA polymerase (2.5 U/ÎŒl) 

3. Prepare the sample reaction(s) as indicated below:<br>Note Stratagene recommends setting up a series of sample reactions using various concentrations of dsDNA template ranging from 5 to 50 ng (e.g., 5, 10, 20, and 50 ng of dsDNA<br>template) while keeping the primer concentration constant. 

5 ÎŒl of 10Ã reaction buffer<br>X ÎŒl (5â50 ng) of dsDNA template<br>X ÎŒl (125 ng) of oligonucleotide primer #1<br>X ÎŒl (125 ng) of oligonucleotide primer #2<br>1 ÎŒl of dNTP mix 

ddH2O to a final volume of 50 ÎŒl 

Then add<br>1 ÎŒl of PfuTurbo DNA polymerase (2.5 U/ÎŒl) 

4. If the thermal cycler to be used does not have a hot-top assembly, overlay each reaction with ~30 ÎŒl of mineral oil.<br>5. Cycle each reaction using the cycling parameters outlined as follow (For the control reaction, use a 5-minute extension time and run the reaction for 18 cycles.) 

95°C 30 seconds, 1 cycle<br>12â18 cycles of 95 °C 30 seconds, 55 °C 60 seconds, 68°C 1min/kb 

<br> 

6. Adjust segment 2 of the cycling parameters in accordance with the type of mutation desired (see the follow):<br>Point mutations 12<br>Single amino acid changes 16<br>Multiple amino acid deletions or insertions 18 

Note: If desired, amplification may be checked by electrophoresis of 10 ÎŒl of the product on a 1% agarose gel. A band may or may not be visualized at this stage. In either case proceed with Dpn I digestion and transformation