User:Mar/Notebook/2007-8-23

Optimization of PCR cycles for genotyping (6, part 3)

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' - 45-65°C/1')x30 - 72°C/10' (annealing every 5°C)
• prepared 6x10µL =< 70µL of mix:

GreenGoTaq - 35µL
3 primers - 3.5µL each
water - 21µL
rl117a (het) DNA - 3.5µL

• aliquoted : a 10µL, added 10µL mineral oil and frozen all tubes on dry ice
• started EP40 and EP45, then frozen
• started EP50 and EP55, then frozen
• started EP42.5 and EP7.5, then frozen
• started EP52.5 and EP57.5, then frozen
• thawed and run gel on 10µL 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

• balanced doublet @ 55°C confirmed, though a little weaker then best unbalanced doublet (55°C)
• annealing at the temp. < 55°C yields gradually weaker doublets, with longer band always weaker than shorter
• annealing at the temp. 60°C yields only longer band
• annealing at the temp. 65°C yields no bands

Future directions

• recheck the same program for annealing temp. range 53 - 54 - 55 - 56 - 57 - 58°C