Yeast colony PCR

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1. Pick part of a colony into a PCR tube using a sterile pipet tip or toothpick. Mark the location of the picked colony with a dot and number on the bottom of the plate so you can go back and pick the rest of it to grow up for a glycerol stock if it is correct.

2. Microwave picked colonies on high for 1 min.

3. For each PCR, prepare the following reaction mix:

   Component	Volume
   10X Taq buffer (NEB M0273L)	5 μL
   10 mM dNTPs (2.5 mM each)	1 μL
   10 μM forward primer	1 μL
   10 μM reverse primer	1 μL
   Taq polymerase (NEB M0273L)	0.5 μL
   H₂O	41.5 μL

4. Add 50 μL PCR mix to each microwaved colony.

5. Cycle using the following parameters:

   Step	Temperature	Time
   Initial denaturation	95C	5’
   Amplification (35 cycles)	95C	30”
   	55C	30”
   	68C	1’/kb
   Final extension	68C	5’
   Hold	10C	∞

6. Add 10 μL 6X loading dye, pipet up and down to mix, and load 10 μL on an agarose gel.