Site Directed Mutagenesis- (Kunkel Method)

 

<center> <h2> <b><font><font>Site Directed Mutagenesis- (Kunkel Method) </font> </font> </b></h2> </center>

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