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Colony PCR in pombe

Forsburg Lab Protocols

Colony PCR for discrimination between homologous and nonhomologous integration of markers in S. pombe

This protocol was shared by shared by Cindy Troxell, Dept Molecular, Cellular and Developmental Biology, University of Colorado, Boulder CO.

Overview.

Colony PCR can be used to identify colonies where your favorite gene (yfg) has been replaced with a marker gene by homologous recombination, and to distinguish homologous recombination events from non-homologous. It can also be used to identify colonies from a tetrad that carry a particular gene replacement. This is not a substitute for Southern blotting, but an adjunct.

Outline of steps:

General Instructions:

1. Making cell suspensions. To 30ul of sterile milliQ H2O (dH2O) add a small glob of S. pombe cells. (Fewer cells is better than too many.) A sterile, pointed-end toothpick works well for transfer. Cell suspensions can be stored at room temperature for a day, or at 4° C for longer periods. Do not boil the suspensions!

2. Making PCR master mix. Make one stock for N reactions, where N = the number of colonies to be tested plus a few extra reactions to account for pipetting error. Multiply vol per reaction by N for total volume to use. For example, for 30ul reactions (final volume):

component vol per rx

10X PCR reaction buffer 3.0 ul

MgCI2, 25mM stock 1.2ul

dNTPs, 10mM stock (2.5 mM each dNTP) 1.2ul

sterile dH2O 11.45ul

sense primer, a, 20uM stock 1.5ul

antisense primer, b or c, 20uM stock 1.5ul

polymerase, 5U/ul stock 0.15ul

3. Assembling and cycling reactions. Invert the eppendorf tube containing the master mix gently until the Taq polymerase is suspended evenly. Aliquot 20ul per 0.5ml reaction tube. Add 10ul of cell suspension to each reaction tube (pipette up & down to resuspend cells first) for a final volume of 30ul. Overlay each reaction with mineral oil if needed. (Cheap mineral oil from teh local pharmacy has worked well for me. Autoclave before use. If it turns cloudy, let it sit a day or two to clear.) Cycle the tubes in your local thermal cycler. Typical cycle steps, for primers with Tm's of ca. 50-60° C, are

30 sec. to 1 min. at 94° C
1.5 min at 43° C
1.5 minat 72° C

30 cycles are adequate, but product has failed to appear with fewer. The first time you use your primers, test for non-specific bands in single primer control reactions on a strain you know to have the marker of interest. Raise the annealing temp. as needed to get rid of any single-primer bands. Always run a no-template reaction, substituting sterile dH2O for cell suspension, as a control for artifactual bands.

4. Analyzing products on an agarose gel. Add 6ul of 6X loading buffer to each PCR reaction and load as much as possible onto a 1XTAE agarose gel. E.g., products from 0.5 to 4 kb are easily resolved on a 2% gel.

This protocol comes from Cindy Troxell at the University of Colorado, Boulder. Questions should be directed to Cindy.

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