Stock No: 008217
Protocol 23570: Standard PCR Assay - Car4<tm1Sly>
Version 2.2

Notes

Fewer than expected homozygotes are produced from heterozygote crosses (with even fewer female homozygotes produced). This from IMR Database.
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

HET = 700 bp and 548 bp
MUT = 700 bp
WT = 548 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR8222 TGT GGA TCT TGA TGG GTT GTT TGT ACA CAG TCC Mutant Reverse A
oIMR8223 TGA GGT CAA TTC CCA GCT CCC ACA TTG TTG C Mutant Forward A
oIMR8970 GGC CTG AGA AGT GGC CTG GA Wild type Forward A
oIMR8971 AGC CCT GTC CTG GGC CTG AT Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR8222 0.50 uM
oIMR8223 0.50 uM
oIMR8970 0.50 uM
oIMR8971 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.