User:Mar/Notebook/2007-8-2
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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:
| AWS | AWS30 | |
|---|---|---|
| 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
