Protocol 29193 - Ass1<tm1Bay> STD PCR

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 = 195 bp
Heterozygote = 279 bp and 195 bp
Wild type= 279 bp

This assay does not work well without the use of a Hotstart Taq polymerase.

Run pcr product on a 1.5% electrophoresis gel.<br /> <br /> Add 2 µl Cresol Red to samples post PCR.

JAX Protocol

Protocol Primers

Primer	Sequence 5' → 3'	Primer Type	Reaction
oIMR9536	CTG CAT AGG CCC AGA CTC TC	Wild type Forward	A
oIMR9537	CAC AGA CCT TCC TCC ATC GT	Common	A
oIMR9538	ATT GCA TCG CAT TGT CTG AG	Mutant Forward	A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTP KAPA	0.26 mM
oIMR9536	0.50 uM
oIMR9537	0.50 uM
oIMR9538	0.50 uM
Glycerol	6.50 %
Dye	1.00 X
Kapa 2G HS taq polym	0.03 U/ul
DNA

Cycling

Step	Temp °C	Time	Note
1	94.0	--
2	94.0	--
3	65.0	--	-0.5 C per cycle decrease
4	68.0	--
5		--	repeat steps 2-4 for 10 cycles (Touchdown)
6	94.0	--
7	60.0	--
8	72.0	--
9		--	repeat steps 6-8 for 28 cycles
10	72.0	--
11	10.0	--	hold

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

JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

This is the only strain that uses this protocol.