2. Dilute Primers to 80-85Ng/Ul (A 1:10 Dilution of 100Um Primer Gives a Close Approximation)

PCR Mutagenesis

1. Thaw reagents on ice.

2. Dilute primers to 80-85ng/ul (A 1:10 dilution of 100uM primer gives a close approximation)

3. In a 0.2ml PCR tube prepare the control reaction as follows:

5 μl of 10× reaction buffer

2 μl (10 ng) of pWhitescript 4.5-kb control plasmid (5 ng/μl)

1.25 μl (125 ng) of oligonucleotide control primer #1

1.25 μl (125 ng) of oligonucleotide control primer #2

1 μl of dNTP mix

39.5 μl of double-distilled water (ddH2O) to a final volume of 50 μl

Then add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl)

4. According to the official protocol, “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 template) while keeping the primer concentration constant.”

If the correct concentration of DNA template to use is unknown, DNA templates should be diluted to approximately 20-25ng/ul and four different volumes used to prepare reaction mixtures (as described in the following step).

4. Prepare 0.2ml PCR tubes for each sample reaction as follows:

5. Run the thermal cycler with the following parameters:

*Use 12 cycles for point mutations, 16 cycles for single amino acid changes, and 18 cycles for multiple amino acid deletions or insertions

6. Add 1 ul of Dpn I restriction enzyme to each amplification reaction and gently mix by pipetting the solution up and down. Spin down the reaction mixtures for one minute in a microcentrifuge and incubate in a dry incubator at 37°C for one hour.

7. Thaw Epicurian Coli XL1-Blue supercompetent cells on ice.For each sample, aliquot 50ul of the cells into pre-chilled 1.5ml tubes.

8. Add 1ul of Dpn I-treated DNA samples to separate aliquots of the supercompetent cells. Gently swirl to mix and incubate the reactions on ice for 30 minutes. While reactions are incubating, pre-warm LB to 42°C.

9. Heat shock the transformation reactions for 45 seconds at 42°C and then place the tubes immediately on ice for 2 minutes.

10. Add 500ul of pre-warmed LB to each of the reactions, place tubes in a rack, tape the tubes down, and place the rack on its side in a 37°C shaker at 225-250 rpm for one hour.

11. While the reactions are incubating, pre-warm LB agar plates containing the appropriate antibiotic for the plasmid vector upside down in a 37°C incubator.

12. Plate the entire volume of the reaction on the pre-warmed plates and incubate them right side up for at least an hour in a 37°C incubator, until the surfaces of the plates are dry, and then turn them upside down in the incubator. Plates should incubate for at least 16 hours.