Stock No: 003248
Protocol 28349: Standard PCR Assay - Il12rb2<tm1Jm>
Version 4.2

Notes

This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase).
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

Melt Peaks
Wt = 84.7°C +/- 1
Mut = 90.7
°C +/- 1


Mutant = 500 bp
Heterozygote = 265 bp and 500 bp
Wild type = 265 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
oIMR0204 CAC GGG TAG CCA ACG CTA TGT C A
oIMR0221 GCC CTG AAT GAA CTG CAG GAC G A
oIMR0799 GTG TGC AAG CTT GGC ACT GTG ACC GTC CAG A
oIMR0800 GTT TAG CTT GCA GAC AAA CAA GGT CAT ACC A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR0204 0.50 uM
oIMR0221 0.50 uM
oIMR0799 0.50 uM
oIMR0800 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
003248 B6;129S1-Il12rb2tm1Jm/J
003642 CBy.129S1-Il12rb2tm1Jm/J
2 strains use this protocol