Genotyping Protocols Database
Protocol 22289: Standard PCR Assay - Tg(HLA-A2.1)1Enge
Version 1.3

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.
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

Transgene = 111 bp
Internal positive control = 200 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
oIMR1332 TTC TCC CTC TCC CAA CCT ATG TAG Transgene A
oIMR4311 CGA CGA CAC TGA TTG GCT TCT Transgene A
oIMR8744 CAA ATG TTG CTT GTC TGG TG Internal Positive Control Forward A
oIMR8745 GTC AGT CGA GTG CAC AGT TT Internal Positive Control Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR1332 0.50 uM
oIMR4311 0.50 uM
oIMR8744 0.50 uM
oIMR8745 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
003475 C57BL/6-Mcph1Tg(HLA-A2.1)1Enge/J
005512 NOD.B6-Mcph1Tg(HLA-A2.1)1Enge/DvsJ
009617 NOD.Cg-Mcph1Tg(HLA-A2.1)1Enge Prkdcscid Il2rgtm1Wjl/SzJ
004262 NOD.Cg-Mcph1Tg(HLA-A2.1)1Enge Prkdcscid/Dvs
014570 NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A/H2-D/B2M)1Dvs/SzJ
5 strains use this protocol