Protocols &Technical Information
Yeast Deletion Database (consortium members only)
Confirmation PCR:
Introduction/ General Strategy
A colony PCR strategy is used to identify successful deletions. This method
is fast, reliable, and amenable to a 96-well format. Many of steps in
the following protocol have been taken from Achim
Wach's application protocol which provides an excellent description
on how to use the kanMX modules.
Four confirmation primers (A, B, C, and D) have been designed for each
ORF. These primers, along with two kanMX4 specific primers (kanB
and kanC), can be used to check both of the novel junctions in a putative
transformant. We generally perform five different PCR tests on each colony
(A-B, C-D, A-kanB, kanC-D, and A-D).
In an unsuccessful deletion, the ORF specific primer pairs (A-B and C-D)
should generate PCR products because the ORF is still present. In contract,
kan specific primer pairs (A-kanB and kanC-D) should not generate PCR
products in an unsuccessful deletion.
In a successful deletion, PCR products should be obtained in the kan specific
reactions (A-kanB and kanC-D) but not in the ORF specific reactions (A-B
and C-D). In addition, the size of the A-D product will most likely change
after the ORF has been replaced by the kanMX4 module (this can
be confirmed by a Hind III digest).
last updated January 2002 amchu@cmgm.stanford.edu
