Genotyping Protocols Database
Stock No: 002288
Protocol 29298: Standard PCR Assay - Ighm<tm1Cgn>
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

Mutant Tm = 88°C
Wild type Tm = 84°C

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 Reverse 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
021871 BXSB.129S2(B6)-Ighmtm1Cgn/DcrJ
9 strains use this protocol