Yeast Colony PCR
Akada et al., Biotechniques 28:668-674 (April 2000)
MATERIALS0.25% 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 72C) 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:
95C 2 minutes
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95C 1 minute
55C 1 minute
72C 2 minutes
==> 30 cycles
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72C 5 minutes
- Load entire sample on agarose gel.
Optional alternative
Quick SDS extraction protocol1. 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).
PCR mixture1. Combine reaction mix on ice:
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)
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==> water to 49 µl
2. Add 0.8 µl of genomic DNA from extraction procedure.
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