User:Mar/Notebook/2007-8-17

Optimization of PCR cycles for genotyping (6)

Goal

• shorten PCR cycling while preserving detection level
• testing 2-step PCR

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.

Acc. to Eppendorf BioNews Application Notes (Lopez & Prezioso, A better way to optimize: Two-step gradient PCR, Sept 2001):

• test cycle: 94°C/2' - (94°C/15" - 50-70.5°C/30")x30 - 72°C/1'
• screen for intensities of specific vs. nonspecific amplicons

Protocol

• cycle: 94°C/5' - (94°C/1' - 50-70.5°C/1')x30 - 72°C/10' (annealing every 2.5°C)
• prepared 11x10µL =< 120µL of mix:

GreenGoTaq - 60µL
3 primers - 6µL each
water - 36µL
rl117a (het) DNA - 6µL

• aliquoted : a 10µL, added 10µL mineral oil and frozen all tubes on dry ice
• started EP50 and EP55, then frozen
• started EP60 and EP65, then frozen
• started EP70 and EP75, then frozen
• started EP52.5 and EP57.5, then frozen
• started EP62.5 and EP67.5, then frozen
• started EP72.5 then frozen
• 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:

	AWS	AWS60	AWS20	AWS10	AWS01
94°C	5'	5'	5'	5'	5'
94°C	1'	1'	20"	10"	1"
55°C	2'	1'	20"	10"	1"
72°C	3'	1'	20"	10"	1"
cycles total	30x	30x	30x	30x	30x
72°C	10'	10'	10'	10'	10'
total time [hr] @ 20µL	3.82	2.32	1.32	1.08	1.00

Results

• no bands, except for a hint in 50°C annealing

Future directions

• run the same program for annealing temp. range 40°C-55°C