Protocol 9509: Separated PCR Assay - Ppm1f<tm1Dgen>

B6.129P2-Ppm1f^tm1Dgen/J

Stock No: 005831

Version 1.0

Notes

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Mutant = 431 bp
Heterozygote = 239 bp and 431 bp
Wild type = 239 bp

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
moIMR0012		GGG TGG GAT TAG ATA AAT GCC TGC TCT		Mutant Forward
oIMR4940		CTA AGC CTA AAC TGC GTG GCT TTA C		Wild type Forward
oIMR4941		AAG CAG GAA GGG ACA CGT GTC GGT C		Common

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.00
MgCl2	2.00
dNTPS-kapa	0.20
Primer 1	0.50
Primer 2	0.50
10x Loading Dye	0.50
Kapa 2G HS taq polym	0.01
DNA

Cycling

Step	Temp °C	Time	Note
1	94.0	--
2	94.0	--
3	65.0	--	-1.5 C per cycle decrease
4	68.0	--
5		--	Go to step 2-4, 10 times
6	94.0	--
7	50.0	--
8	72.0	--
9		--	Go to step 6-8, 28 times
10	72.0	--
11	10.0	--	hold

JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Reaction B

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.00
MgCl2	2.00
dNTPS-kapa	0.20
Primer 3	0.50
Primer 4	0.50
10x Loading Dye	0.50
Kapa 2G HS taq polym	0.01
DNA

Cycling

Step	Temp °C	Time	Note
1	94.0	--
2	94.0	--
3	65.0	--	-1.5 C per cycle decrease
4	68.0	--
5		--	Go to Step 2-4, 10 times
6	94.0	--
7	50.0	--
8	72.0	--
9		--	Go to Step 6-8, 28 times
10	72.0	--
11	10.0	--	hold

JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.