2/7 PCR & isolate DNA
2/7 PCR & isolate DNA kdorfman Wed, 01/18/2012 - 20:38Set 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??