ADE 2 Transformation Control
ADE2 serves as a good control for transformations because targeted deletions
result in red colonies which are easy to score. The targeting efficiency
(correct deletions/total) can be determined by measuring the percentage
of red colonies. The standard two-step PCR procedure is used to generate
this control construct.
ADE2 Primer Sequences
Three different primers are required to generate the ADE2 deletion construct
(click here to see an outline of the PCR
- This 56mer primer contains (5' --> 3'): common tag priming site
(18 bases) , tag sequence (20 bases- green),
homology to the 5'-side of the kanMX4 module (18 bases-lower
ADE2 UP-HOMOLOGY 63mer:
- This 63mer contains (5' --> 3'): 45 bases of upstream homology
(coding strand on the 5'-side of the ADE 2 ORF, the start codon is in
red), 18 bases homologous to the common
tag priming site (lower case).
ADE2 DOWN-HOMOLGY 64mer:
- This 63mer contains (5' --> 3'): 45 bases of downstream homology
(non-coding strand on the 3'-side of the ADE 2 ORF), 19 bases homologous
to the 3'-side of the kanMX4 module (lower case).