One-Step Deletion/Tagging

One Step Gene Disruption/Tagging

Materials

• Appropriate primers
• Appropriate template plasmid (pFA6 series)
• dNTPs
• Taq DNA polymerase and Buffer

Method

• Prepare the following Master Mix:

Reagent Volume F1 or F2 Primer 5 ?l R1 Primer 5 ?l 2 mM dNTPs 50 ?l 10X Taq Buffer 50 ?l pFA6 pl.templ. (~250ng) 5 ?l H2O 382.5 ?l Taq 2.5 ?l

In case of NATMX cassettes add 5% DMSO

• Mix and aliquote 100 ?l in each 0.2 ml PCR eppendorf tube.
• Run the following PCR program:

PCR program 2' 95°C 5 cycles 1' 94°C 1' 45°C 4' 72°C 30 cycles 1' 94°C 1' 52°C 4' 72°C 10' 72°C