Protocols &Technical Information
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.
- Sequences for the ADE2 deletion primers (see below)
- pFA-kanMX4 plasmid (contact the Philippsen lab)
- ADE 2 PCR protocol
ADE2 Primer Sequences
Three different primers are required to generate the ADE2 deletion construct
(click here to see an outline of the PCR
deletion strategy).
ADE2TAG 56mer:
5'GATGTCCACGAGGTCTCTTTGGTGCGCCCACAAACAAAcgtacgctgcaggtcgac3'
- 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 case).
ADE2 UP-HOMOLOGY 63mer:
5'AACAATCAAGAAAAACAAGAAAATCGGACAAAACAATCAAGTATGgatgtccacgaggtctct3'
- 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:
5'TTATAATTATTTGCTGTACAAGTATATCAATAAACTTATATATTAatcgatgaattcgagctcg3'
- 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).