2/7 PCR & isolate DNA

Set up PCR to verify plasmid inserts

Isolate plasmid DNA from bacteria

Outside class: Begin collecting virgin females from the P- element lines this week

PCR reagents

  • Invitrogen T7 Promoter Primer Catalog Number N560-02 (forward primer, 20uM)
  • 5 µL sterile water
  • PCR buffer
  • 50 mM MgCl2
  • 10 mM dNTP mix
  • Taq polymerase (5U/µL)

PCR engine parameters

  • 94 C 5 min
  • 94 C 30 sec
  • 55 C 30 sec
  • 72 C 3 min
  • repeat 30 x
  • 72 C 6 min
  • (protocol says 4C, should just be END) Note - can just stop, cold promotes condensation, and the product is sterile

DNA Isolation reagents

  • Solution 1 (~7.2 mL minimum)
    • 50 mM glucose
    • 10 mM EDTA
    • 25 mM Tris, pH 8
  • Solution 2 (14.4 mL minimum)
    • 0.2 M NaOH
    • 1% SDS *Solution 3 (~10.8 mL minimum)
    • 2.7 M KoAc
    • 6.6 M acetic acid
  • 100% EtOH
  • 80% EtOH
  • sterile water

Speed Vac??