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Molecular Biology Lab Manual - Site Directed Mutagenesis- (Kunkel Method)- appendix page 15

Site Directed Mutagenesis- (Kunkel Method)

1. Phosphorylate mutagenic oligonucleotide.

Incubate at 37oC for 45 min.

2. Second strand synthesis.
Annealing Heat to 95oC for 3 - 5 minutes, cool to 4oC slowly over 20 - 30 minutes.
Centrifuge briefly to bring contents to bottom of tube. Place on ice.

To the annealed template-primer, add:
Incubate on ice for 5 min., at room temp for 5 min., then at 37oC overnight.

If impatient, 5 uL may be removed after 2 hours and used for transformation. Otherwise, transform 5 uL into an appropriate strain (ung+) after the overnight incubation.

10X T4 Kinase Buffer

5X Synthesis Buffer

10X Annealing Buffer

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