Stock No: 002189
Protocol 29193: Standard PCR Assay - Ass1<tm1Bay> STD PCR
Version 4.2

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 &micro;l Cresol Red to samples post PCR.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
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.