User:Mar/Notebook/2007-8-2

= Optimization of PCR cycles for genotyping (1) = Goal: shorten PCR cycling while preserving detection level

GreenGoTaq - 40µL primers - 4µL each water - 24µL rl117a (het) DNA - 4µL
 * prepared 4x20µL = 80µL of mix:
 * 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:

= 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