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![]() | Gene Disruption via PCR[Adapted from Brachmann et al. Yeast, 14:115-132 (1998).]Reaction mix:5 µl 10X Taq Buffer 5 µl 25 mM MgCl2 2 µl 10 mM dNTP's 10-100 ng template DNA 25 pmols of each primer 0.5 µl Taq polymerase (2 Units) ____________________ ==> water to 50 µl total volume PCR cycle profile: 94C 5 minutes _________________ 94C 1 minute 55C 1 minute 72C 2 minutes ==> 10 cycles _________________ 94C 1 minute 65C 1 minute 72C 2 minutes ==> 20 cycles _________________ 72C 10 minutes NOTE: The entire reaction can be used for a transformation without any further purification. MATERIALS 10X Taq Buffer: 0.5 M KCl 100 mM Tris-Cl, pH 8.5 1% Triton X-100 25 mM MgCl2 10 mM dNTP's pRS40X template DNA - mini-prep DNA works well Taq polymerase Two Gene-specific DNA primers: One oligonucleotide should consist of 40 nts of gene-specific sequence for one end of the targeted region at the 5' end followed by: 5'-CTGTGCGGTATTTCACACCG-3' (left primer), and another 40 nt homologous to the other side of the targeted region at the 5' end followed by: 5' AGATTGTACTGAGAGTGCAC-3' (right primer). The primers are then used to amplify any auxotrophic marker from a pRS40X or pRS30X integrating plasmid (Sikorski & Hieter, 1989; Brachmann et al. 1998).
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