Protocol 19783: Standard PCR Assay - Tg(HLA-A/H2-D)2Enge
Version 1.0

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 = 356 bp
Heterozygote = 283 bp and 356 bp
Wild type = 283 bp

This assay is able to distinguish tg/o from tg/tg.  Integration site is known to be on mChr 12.

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
29239 AGT TGT GCT GAT GCA ACT GG Common A
29240 TCA GTC TCA AAC GGC TTT CA Wild type Reverse A
29241 TTA ACA TCT TGA GCT AGG GCT GA Transgene Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
29239 0.50 uM
29240 0.50 uM
29241 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
004191 B6.Cg-Immp2lTg(HLA-A/H2-D)2Enge/J
006604 NOD/ShiLtDvs-Tg(HLA-A/H2-D/B2M)1Dvs/J
2 strains use this protocol