Yeast Colony PCR
Akada et al., Biotechniques 28:668-674 (April 2000)
MATERIALS
- 0.25% SDS
- 10X Colony PCR Buffer:
- 0.125 M Tris-HCl pH 8.5
- 0.5625 M KCl
- 25 mM MgCl2
- 10 mM dNTP's
- 20% Triton X-100
- Taq polymerase
- Two Gene-specific DNA primers:
- Each oligonucleotide should be 25 nucleotides long and specific for either side of the region of interest to be amplified.
NOTE:
The elongation times (at 72°C) work well for amplification of loci <1.5 kbp in size. These conditions may need to be modified for amplification of longer regions.
The elongation times (at 72°C) work well for amplification of loci <1.5 kbp in size. These conditions may need to be modified for amplification of longer regions.
PCR mixture
-
Combine reaction mix on ice:
2.5 µl 10X Colony PCR Buffer
1.5 µl 25 mM MgCl2
0.5 µl 10 mM dNTP's
10 pmols of each primer
1.25 µl 20% Triton X-100
0.25 µl Taq polymerase (5 Units/µl)
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==> water to 24 µl - Use pipette tip to transfer yeast cells (just enough to cloud the reaction) into the tubes.
- PCR cycle profile:
95°C 2 minutes
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95°C 1 minute
55°C 1 minute
72°C 2 minutes
==> 30 cycles
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72°C 5 minutes
- Load entire sample on agarose gel.
Optional alternative
Quick SDS extraction protocol
1. Prepare microfuge tubes containing 20 µl 0.25% SDS.
2. Use pipette tip to transfer yeast cells (about 1E7) into the tubes. Vortex 10 sec., heat at 90°C 3 min, and centrifuge for 30 sec.
3. Add 0.8 µl of supernatant into PCR mixture (see below).
2. Use pipette tip to transfer yeast cells (about 1E7) into the tubes. Vortex 10 sec., heat at 90°C 3 min, and centrifuge for 30 sec.
3. Add 0.8 µl of supernatant into PCR mixture (see below).
PCR mixture
1. Combine reaction mix on ice:
2. Add 0.8 µl of genomic DNA from extraction procedure.
5 µl 10X Colony PCR Buffer
3 µl 25 mM MgCl2
1 µl 10 mM dNTP's
20 pmols of each primer
2.5 µl 20% Triton X-100
0.5 µl Taq polymerase (5 Units/µl)
____________________________
==> water to 49 µl
3 µl 25 mM MgCl2
1 µl 10 mM dNTP's
20 pmols of each primer
2.5 µl 20% Triton X-100
0.5 µl Taq polymerase (5 Units/µl)
____________________________
==> water to 49 µl
2. Add 0.8 µl of genomic DNA from extraction procedure.
