| Contributor: |
The Laboratory of Jasper Rine at the University of California, Berkeley
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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) (see Hint #1).
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| Template DNA |
| Sikorski and Hieter vectors, pRS40... series which contain selectable marker genes (i.e. HIS3, URA3, LEU2, etc.)
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| Oligonucleotide Primer 2 |
| 40 nucleotides homologous to the other side of the targeted region at the 5'end followed by 5'-AGATTGTACTGAGAGTGCAC-3'
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| Oligonucleotide Primer 1 |
| 40 nucleotides of gene-specific sequence at the 5' end followed by 5'-CTGTGCGGTATTTCACACCG-3'
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| 10 mM dNTPs |
| 10 mM dATP
10 mM dTTP
10 mM dGTP
10 mM dCTP
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| 25 mM MgCl2 |
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| 10X Taq Buffer |
| 100 mM Tris-Cl, pH 8.5
0.5 M KCl
0.1% Triton-X 100
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Oligonucleotide
Triton X-100
dCTP
dTTP
dGTP
Tris
dATP
Potassium Chloride
Magnesium Chloride
Vector
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1. Purifying the PCR product increases the transformation efficiency dramatically.
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2. Sikorski, R.S. and P. Hieter. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics1989; 122: 19-27.
1. Brachmann, C.B., A. Davies, G.J. Cost, E. Caputo, J. Li, P. Hieter, and J.D. Boeke (1997). Designer deletion strains derived from Saccharomyces cerevisiae s288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast (In press).
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