Stock No: 004144
Protocol 23042: Standard PCR Assay - Nr1h4<tm1Gonz>
Version 2.3

Notes

A 1989 bp read-through band may be amplified from the mutant allele.
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 = 291 bp
Heterozygote = 249 bp and 291 bp
Wild type = 249 bp
<p>Separated by gel electrophoresis on a 3% agarose gel.</p>

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR4056 TCT CTT TAA GTG ATG ACG GGA ATC T Wild type Forward A
oIMR4057 GCT CTA AGG AGA GTC ACT TGT GCA Mutant Forward A
oIMR4058 GCA TGC TCT GTT CAT AAA CGC CAT Common A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR4056 0.50 uM
oIMR4057 0.50 uM
oIMR4058 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

Stock Number Strain Name
007214 B6.129X1(FVB)-Nr1h4tm1Gonz/J
004144 STOCK Nr1h4tm1Gonz/J
2 strains use this protocol