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Optimization of PCR cycles for genotyping (1)

Goal: shorten PCR cycling while preserving detection level

  • prepared 4x20µL = 80µL of mix:
GreenGoTaq - 40µL
primers - 4µL each
water - 24µL
rl117a (het) DNA - 4µL
  • aliquoted : 2x5µL; 2x10µL; 2x20µL, added 10µL mineral oil to 5µL and 10µL tubes and frozen all tubes on dry ice
  • started AWS30 on one set of tubes, then frozen until next day
  • started AWS with another set of tubes o/n, then frozen in the morning
  • (next day) thawed and run gel

ramp time for PTC-200: up to 3°C/sec

programs used:

94°C 5' 5'
94°C 1' 30"
55°C 2' 30"
72°C 3' 30"
cycles total 30x 30x
72°C 10' 10'
total time [min] 181' 46'

Meeting with GM and BD about protein fractionation/purification

Tuesday ferret experiment

  • Experiment is scheduled to proceed as usually (Centricons and beads), with the exception that we use suspension design.
  • Possibly use Oregon Green on suspended GE cells
  • Use whole extract as a positive control

Methodological considerations

  • Extraction: Tris alone vs. Tris + protease inhibitors (could tell whether it's a protein)
  • Purification: crude extracts vs. Acetone precipitated extract (will get rid of lipids and nucleic acids)
  • Protein inactivation: Heat-denaturation and/or Proteinase K digestion (will confirm whether it's a protein)

Warning: Centricons are a considerable source of protein loss