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