Yeast Colony PCR

 

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 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

1. 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
    
2. Use pipette tip to transfer yeast cells (just enough to cloud the reaction) into the tubes.
3. PCR cycle profile:
   
   95C 2 minutes
   _________________
   95C 1 minute
   55C 1 minute
   72C 2 minutes
   ==> 30 cycles
   _________________
   72C 5 minutes
    
4. 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 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:

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.