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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 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).