Genotyping Protocols Database
Protocol 23837: Standard PCR Assay - Il2rg<tm1Wjl>
Version 4.2

Notes

The Il2rg gene is X-LINKED. Males with the targeted allele are hemizygous mutants
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 = ~349 bp
Heterozygote = ~269 bp and ~349 bp
Wild type = ~269 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
oIMR5330 GTG GGT AGC CAG CTC TTC AG Common A
oIMR5331 CCT GGA GCT GGA CAA CAA AT Wild type Reverse A
oIMR7415 GCC AGA GGC CAC TTG TGT AG Mutant Reverse A PGK

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR5330 0.50 uM
oIMR5331 0.50 uM
oIMR7415 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
003174 B6.129S4-Il2rgtm1Wjl/J
003169 C.129S4-Il2rgtm1Wjl/J
010636 NOD.Cg-B2mtm1Unc Prkdcscid Il2rgtm1Wjl/SzJ
009617 NOD.Cg-Mcph1Tg(HLA-A2.1)1Enge Prkdcscid Il2rgtm1Wjl/SzJ
023848 NOD.Cg-Prkdcscid H2-K1tm1Bpe H2-D1tm1Bpe Il2rgtm1Wjl/SzJ
005557 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ
007799 NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ
7 strains use this protocol