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Tuesday, November 18, 2003
Description Yeast Colony PCR Procedure 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) ____________________________ ==> 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 _________________ 95C 1 minute 55C 1 minute 72C 2 minutes ==> 30 cycles _________________ 72C 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 90C 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: 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 2. Add 0.8 μl of genomic DNA from extraction procedure. -------------------------------------------------------------------------------- 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 72C) work well for amplification of loci <1.5 kbp in size. These conditions may need to be modified for amplification of longer regions. Recipes Supplies Tips (责任编辑：泉水) |
Yeast Colony PCR
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin 点击:
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