Genotyping Protocols Database
Protocol 29314: Standard PCR Assay - Tg(TcraBDC2.5)1Doi, Tg(TcrbBDC2.5)2Doi
Version 3.3

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

TG/O TG Tm = 86 Area = 10
TG/O WT Tm = 82 Area=2
WT Area =10
WT Tm = 82

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR3844 CAT GTT TCC CTG CAC ATC AG Transgene A
oIMR3845 CCA GAT CCA AAG ATG AGT TGC 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
oIMR3844 0.50 uM
oIMR3845 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
004460 NOD.Cg-Tg(TcraBDC2.5,TcrbBDC2.5)1Doi/DoiJ
024476 NOD.Cg-Stat4tm1Gru Thy1a Ifngr1tm1Agt Tg(TcraBDC2.5,TcrbBDC2.5)1Doi/LmbrJ
2 strains use this protocol