Gene Disruption via PCR
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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)
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==> water to 50 µl total volume
PCR cycle profile:
94C 5 minutes
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94C 1 minute
55C 1 minute
72C 2 minutes
==> 10 cycles
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94C 1 minute
65C 1 minute
72C 2 minutes
==> 20 cycles
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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).
