Night before:

clear vials of wildtype Canton-S, put at 18C for virgins in the morning

12 vials WT Canton-S

12 vials of mutant, mapping stock flies

Get fly nap from EH&S

Try sharing funnel apparatus, with fly nap on the cotton in the Coplin jar

• 12 plates N2 normal worms
• 1 plate each mutation
• alcohol lamps
• at least 12 picks (from Dave)

Start overnight of RNAi plasmid containing bacteria:

• pL4440 empty
• pL4440 + lsy-2
• pL4440 + choice

Plates of bacteria with:

• pL4440 empty
• pL4440 + lsy-2 (chemo assay)

Groups will choose one of the following for phenotypic analysis:

• pL4440 + bli-1
• pL4440 + dpy-5
• pL4440 + unc-22 (from Chase Lab)

10 mL per overnight of sterile LB + 12.5 µg/mL tetracycline

Adding 10 µL of 50 mg/mL ampilicillin stock

• 15 mL conical tubes for overnight, 3 per group, so 36 tubes = 360 mL of broth minimum
• Sterile toothpicks

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??