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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.

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)

Incubate at 37oC for 45 min.

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

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:

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

Site Directed Mutagenesis- (Kunkel Method)

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Site Directed Mutagenesis- (Kunkel Method)

Back to:

Molecular Biology Home Page Jeff Newman's Home Page Biology Department Home Page Lycoming College Home Page

Molecular Biology Home Page
Jeff Newman's Home Page
Biology Department Home Page
Lycoming College Home Page