Site Directed Mutagenesis- (Kunkel Method)
1. Phosphorylate mutagenic oligonucleotide.
- 6 uL Primer [10 pmoles (=100 ng for 33mer)/uL]
- 1 uL 20 mM ATP
- 1 uL 10X T4 Kinase buffer
- 2 uL T4 Kinase (20 units)
2. Second strand synthesis.
Annealing
- X uL Uridine containing ssDNA (1 ug)
- 2 uL phosphorylated primer (120 ng)
- 1 uL 10X annealing buffer
- H2O to 10 uL
Centrifuge briefly to bring contents to bottom of tube. Place on ice.
To the annealed template-primer, add:
- 4.2 uL 5X synthesis buffer
- 1 uL 20 mM ATP
- 2 uL T4 DNA polymerase (5 units)
- 4 uL T4 DNA ligase (4 units)
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
- 0.5 M Tris pH 7.6
- 0.1 M MgCl2
- 50 mM DTT
- 1 mM EDTA
- 1 mM Spermidine HCl
5X Synthesis Buffer
- 20 mM Tris pH 7.5
- 5 mM MgCl2
- 1.5 mM DTT
- 1 mM ATP
- 0.5 mM each dNTP
10X Annealing Buffer
- 0.1 M Tris pH 7.5
- 20 mM MgCl2
- 0.5 M NaCl
