Endogenous tagging of yeast genes

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Notes

-This protocol can be used with any pFA6a-based tagging vector and the F2/R1 primer pairs used to construct the yeast GFP collection.

-For small epitope tags (i.e. 3xFLAG, 3xHA), a 45” extension time works well. For large tags (i.e. AID, 3xFLAG-MNase), a 1’15” extension time is needed.

Protocol

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

   Component	Volume
   5X Q5 buffer	10 μL
   10 mM dNTPs (2.5 mM each)	1 μL
   10 μM F2 primer	2.5 μL
   10 μM R1 primer	2.5 μL
   1-5 ng DNA	X μL
   Q5 polymerase	0.5 μL
   H₂O	to 50 μL

2. Cycle using the following parameters:

   Step	Temperature	Time
   Initial denaturation	98C	30”
   Amplification (25 cycles)	98C	10”
   	55C	30”
   	72C	45” or 1’15”
   Final extension	72C	2’
   Hold	10C	∞

3. Analyze 5 μL of the reaction on an agarose gel.