User:Mar/Notebook/2007-8-13

= Optimization of PCR cycles for genotyping (4) = Goal: shorten PCR cycling while preserving detection level

Technical considerations
Acc. to Eppendorf's Tech Support pages:
 * denaturation: min 1" (for 20µL) or 5" (for 50µL)
 * annealing: 10-20" usually adequate
 * extension: ~50/sec which yields 8sec for reeler, but keep in mind that during annealing (at 55°C), Taq extends too, ith ~25/sec speed.

Protocol
GreenGoTaq - 50µL 3 primers - 5µL each water - 30µL rl117a (het) DNA - 5µL
 * prepared 9x10µL =< 100µL of mix:
 * aliquoted : a 10µL, added 10µL mineral oil and frozen all tubes on dry ice
 * started AWS20 @ 55°C, and AWS20 @ 52.5°C, then frozen
 * started AWS10 @ 55°C, and AWS10 @ 52.5°C, then frozen
 * started AWS01 @ 55°C, and AWS01 @ 52.5°C, then frozen
 * started AWS20 @ 57.5°C, and AWS10 @ 57.5°C, then frozen (deviation: both were run on AWS10 @ 57.5°C)
 * started AWS01 @ 57.5°C o/n, then frozen
 * (next day) thawed and run gel on 5µL loads

ramp time for PTC-200: up to 3°C/sec (actual time @ 20µL: 1°C/sec)

programs used:

Results

 * yield decreases with cycle time increase
 * AWS20: max yield at 55°C, but at 52.5°C bands are more balanced
 * AWS10: max yield at 52.5°C, at higher annealing temp. shorter band disappears
 * 55°C: AWS20 >> AWS10
 * 52.5°C: AWS20 < AWS10

Future directions

 * run {52.5°C vs. 55°C} x {AWS20 vs. AWS10} in duplicates

= Mentoring = Taught thick sectioning to John