Genotyping

• 10.0 ul Ligated genomic DNA (~1/15 fly)
• 2.0 ul 2mM each dNTP
• 1.0 ul 10uM forward primer,* Plac4*
• 1.0 ul 10uM reverse primer, *Plac1 *
• 5.0 ul 10X Taq buffer
• 31.0 ul ddH20
• 0.4 ul 2 units Taq

Perform thermal cycling for recovery of 5' flanking as follows:

Cycle Temp Time # cycles

94C 3 min 1

• Denature 94C 30 sec
• Anneal 60C 1 min 35
• Extend 68C 2 min

72C 10 min 1 4C HOLD

PCR Reaction to amplify 3' Flanking Sequence

• 10.0 ul Ligated genomic DNA (~1/15 fly)
• 2.0 ul 2mM each dNTP
• 1.0 ul 10uM forward primer, Pry4
• 1.0 ul 10uM reverse primer, *Plw3-1 *
• 5.0 ul 10X Taq buffer
• 31.0 ul ddH20
• 0.4 ul 2 units Taq

Perform thermal cycling for recovery of 5' flanking as follows:

Cycle Temp Time # cycles

94C 3 min 1

• Denature 94C 30 sec
• Anneal 55C 1 min 35
• Extend 68C 2 min

72C 10 min 1 4C HOLD