PCR Protocol for PCR-Mediated Gene Disruption

Procedure

1. Set up the following PCR tube:

5 μl of 10X Taq Buffer

5 μl of 25 mM MgCl2

2 μl of 10 mM dNTPs

10 to 100 ng of template DNA

25 pmol of specific primer 1

25 pmol of specific primer 2

0.5 μl of Taq DNA polymerase (2 Units)

ddH2O to 50 μl.

2. Place the tube in the thermocycler and program to run the following PCR profile:

94°C for 5 min.

Then 10 cycles of:

94°C for 1 min.

55°C for 1 min.

72°C for 2 min.

Followed by 20 cycles of:

94°C for 1 min.

65°C for 1 min.

72°C for 2 min.

Then followed by 72°C for 10 min.

3. Transform yeast with the entire reaction (see Protocol on Transformation of Yeast with DNA)

Solutions

Template DNA Sikorski and Hieter vectors, pRS40. series which contain selectable marker genes (i.e. HIS3, URA3, LEU2, etc.)

Oligonucleotide Primer 2 40 nucleotides homologous to the other side of the targeted region at the 5'end followed by 5'-AGATTGTACTGAGAGTGCAC-3'

Oligonucleotide Primer 1 40 nucleotides of gene-specific sequence at the 5' end followed by 5'-CTGTGCGGTATTTCACACCG-3'

10 mM dNTPs 10 mM dATP

10 mM dTTP

10 mM dCTP

10 mM dGTP

25 mM MgCl2

10X Taq Buffer 100 mM Tris-Cl, pH 8.5

0.5 M KCl

0.1% Triton-X 100