(Hahn lab) 9/17/02

This method is more reliable than the old method of adding yeast directly to PCR tubes. No more than 1 microliter of the crude DNA should be added to the 50 microliter PCR reaction because the SDS in the DNA prep can inhibit the PCR reaction.

Note: These elongation times and annealing temperatures work well for most applications where the product is less than 1.0 kb. These parameters may have to be adjusted for your specific application .

Platinum Taq (or other hot start Taq) gives more reliable and consistent results than regular Taq for this assay.

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1. Prepare Yeast DNA (this works best with fresh yeast plates)

Use a pipetman tip to transfer the equivalent of a medium size yeast colony to 30 microliters of 0.2% SDS

Vortex ~15 seconds

Heat in hot block for 4 min at 90 deg. (important for consistent DNA extraction and PCR results)

Spin in microfuge 1 min. Remove supernatant to a new tube. The crude DNA can be stored at -20 degrees.

2. PCR Reaction

Combine the following components on ice:

5 microliters 10x colony PCR buffer
1.5 microliters 50 mM MgCl2
1 microliter 10 mM mix of dNTPs
10 pmoles of each primer (~100 ng of a 25 mer oligo)
2 microliters 25% Triton X-100
0.3 microliter Platinum Taq Polymerase [Invitrogen] (5 u/microliter)
H2O to a final volume of 49 microliters

Add mix to PCR tubes on ice containing 1 microliter of the crude DNA prep from above

3. PCR cycle profile:

95 deg 1 min
95 deg 30 sec
54 deg 1 min (optimum annealing temp varies according to primiers)
72 deg 1 min
repeat steps 2 through 4 for a total of 35 cycles
72 deg 6 min
hold at 4 deg

Load 5-10 microliters to Agarose gel for assay.

For DNA sequencing analysis of product, purify using QIAquick PCR purification kit (Qiagen), eluting the product in 30 microliters. Use ~6 microliters for DNA sequencing analysis.

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MATERIALS

0.2% SDS

10X Colony PCR buffer:
0.13 M Tris-HCl pH 8.5
0.56 M KCl

50 mM MgCl2

10 mM dNTP mix

25% Triton X-100

PCR primers ~25 bases in length specific for the region of interest with annealing temp 55-60 deg.

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