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
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72C 5 minutes


Load entire sample on agarose gel.



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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.


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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.


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