Stock No: 002286
Protocol 29018: Standard PCR Assay - Ifng<tm1Ts>
Version 10.2

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 = ~500 bp
Heterozygote = ~210 bp and ~500 bp
Wild type = ~210 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
oIMR6218 CCT TCT ATC GCC TTC TTG ACG Mutant Forward A Neo F
oIMR8284 AGA AGT AAG TGG AAG GGC CCA GAA G Wild type Forward A
oIMR8285 AGG GAA ACT GGG AGA GGA GAA ATA T Common A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR6218 0.50 uM
oIMR8284 0.50 uM
oIMR8285 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
002287 B6.129S7-Ifngtm1Ts/J
002286 C.129S7(B6)-Ifngtm1Ts/J
008228 C3.129S7(B6)-Ifngtm1Ts/J
002575 NOD.129S7(B6)-Ifngtm1Ts/DvsJ
4 strains use this protocol