Genotyping Protocols Database
Stock No: 002249
Protocol 22248: Standard PCR Assay - Ighm<tm1Cgn>
Version 1.3

Notes

A ~1500 bp read-through band may be amplified from the mutant allele.
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
oIMR1750 CCG TCT AGC TTG AGC TAT TAG G Common A
oIMR1751 GAA GAG GAC GAT GAA GGT GG Wild type A
oIMR5255 TTG TGC CCA GTC ATA GCC GAA T Mutant Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR1750 0.50 uM
oIMR1751 0.50 uM
oIMR5255 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
002249 B10.129S2(B6)-Ighmtm1Cgn/J
002288 B6.129S2-Ighmtm1Cgn/J
003903 NOD.129S2(B6)-Ighmtm1Cgn/Dvs
004349 C.129S2-Ighmtm1Cgn/J
004639 NOD.129S2(B6)-Ighmtm1Cgn/DoiJ
005306 NODCaj.Cg-Ighmtm1Cgn Tg(Igh-VB1-8/Igh-6m)1Mjsk/FswJ
005309 NODCaj.Cg-Ighmtm1Cgn Tg(Igh-VB1-8/Igh-6)2Mjsk/FswJ
006608 NOD.Cg-Ighmtm1Cgn Tg(IghelMD4)4Ccg/DvsJ
8 strains use this protocol