Yeast Colony PCR

Yeast Colony PCR

adapted from www.fhcrc.org/labs/hahn/methods/mol_bio_meth/pcr_yeast_colony.html

Chris Tachibana May 2004, updated July 2005

• 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.

• Works best if the product is less than 1.0 kb.

• Use Platinum Taq, or other hot start Taq. (As of 2005, we are mostly using Phusion Polymerase)

• Start by choosing a DNA prep method: Method 1 makes a small genomic DNA prep that can be used for several reactions and stored; Method 2 lyses the cells right in the PCR reaction.

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Method 1:

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

• Use a pipetman tip to transfer yeast cells (about this much: • ) to 30 microliters of 0.2% SDS. JuanJo had success using a pellet of 3 x 10⁶ cells from a liquid culture.

• Vortex ~15 seconds

• Heat in hot block for 4 min at 90 deg.

• Spin in microfuge 1 min. Remove supernatant to a new tube. Store at -20 degrees.

2. PCR Mix

PCR can be done in 50µl or 20µl volumes. Either way, use as little DNA as possible (0.5µl). It works best with the Phusion polymerase but you can also do it with Platinum Taq polymerase.

a) Phusion 50µl per tube

• 10 µl 5X buffer HF

• 34 µl water (for 2 primers)

32 µl water (for 3 primers)

• 2 µl each primer from a 5µM stock

• 1 µl 10mM dNTPs

• 0.5 µl Phusion Enzyme

49.5 µl per tube + 0.5µl DNA, use the "Col-Phu" program in the MJResearch PCR machines

b) Phusion 20µl per tube

• 4 µl 5X buffer HF

• 13.3 µl water (for 2 primers)

12.5 µl water (for 3 primers)

• 0.8 µl each primer from a 5µM stock

• 0.4 µl 10mM dNTPs

• 0.2 µl Phusion Enzyme

19.5 µl per tube + 0.5µl DNA (or less), use the "Col-Phu" program in the MJResearch PCR machines

c) Platinum Taq (50µl reactions)

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 of a 5 µM stock)

2 microliters 25% Triton X-100

0.3 microliter Platinum Taq Polymerase [Invitrogen] (5 u/microliter)

H2O to a final volume of 49 microliters (35.2 microliters)

Aliquot 49 µl into PCR tubes.

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

Go to "PCR cycle profile" below.

Method 2:

1. PCR Mix

Combine the following components on ice (per 50 µl reaction):

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 of a 5 µM stock)

2 microliters 25% Triton X-100

H2O to a final volume of 49 microliters (35.5 microliters)

Aliquot 49 µl into PCR tubes.

Touch a yeast colony with a sterile 20µl pipet tip and put the cells directly into a PCR tube with PCR mix. The mix should be slightly cloudy but not dense with cells.

Heat 8 minutes, 94 degrees (program 97 in the Robocycler). While they are heating, dilute the Taq polymerase.

Add 1µl of Platinum Taq Polymerase diluted 1:2 in 1X colony PCR buffer. You can do this without removing the tubes from the Robocycler.

Go to "PCR cycle profile" below.

3. PCR profile

a) for Phusion Taq

98 deg 30 sec

98 deg 5 sec

52 deg 10 sec

72 deg 15 sec

repeat steps 2-4 for 34 additional cycles

72 deg 5 min

4 deg hold

b)for Platinum Taq

95 deg 1 min

95 deg 30 sec

52 deg 1 min (optimum annealing temp varies according to primiers)

72 deg 2 min

repeat steps 2 through 4 for a total of 35 cycles

72 deg 6 min

hold at 4 deg

Load 20µl 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 (if using Platinum Taq)

0.13 M Tris-HCl pH 8.5 87µl of 1.5 M

0.56 M KCl 187 µl of 3 M

726 µl of water

50 mM MgCl2

10 mM dNTP mix

25% Triton X-100 (if using Platinum Taq)

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