Genetics schedule

2012 schedule

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1/24 - Flies

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

1/31 Worms

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

2/6 (M)

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

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??