Genotyping Protocols Database
Stock No: 002420
Protocol 29312: Standard PCR Assay - B2m<tm1Unc>
Version 5.3

Notes

Melting curve analysis is done using a Roche Light Cycler 480.
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 = 88.7°C +/- 1.0°C
Wild type Tm = 83.1°C+/- 1.0°C

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR0160 TCT GGA CGA AGA GCA TCA GGG Mutant Forward A neo
oIMR0184 TAT CAG TCT CAG TGG GGG TG Common A
oIMR0185 CTG AGC TCT GTT TTC GTC TG Wild type Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR0160 0.50 uM
oIMR0184 0.50 uM
oIMR0185 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
002087 B6.129P2-B2mtm1Unc/DcrJ
002309 NOD.129P2(B6)-B2mtm1Unc/J
002420 C.129P2(B6)-B2mtm1Unc/J
002570 NOD.Cg-Prkdcscid B2mtm1Unc/J
004548 NOD.Cg-B2mtm1Unc Mcph1Tg(HLA-A2.1)1Enge Tg(B2M)55Hpl/DvsJ
005356 NOD.129(B6)-B2mtm1Unc Ciitatm1Ccum/BhsJ
006611 NOD.Cg-B2mtm1Unc Tg(HLA-A/H2-D/B2M)1Dvs/DvsJ
008542 NOD.Cg-B2mtm1Unc Tg(GFAP-B2m)9Mdos/J
010565 NOD.129P2(B6)-B2mtm1Unc Tg(HLA-A24/H2-D/B2M)3Dvs/J
010636 NOD.Cg-B2mtm1Unc Prkdcscid Il2rgtm1Wjl/SzJ
018907 NOD.Cg-B2mtm1Unc Tg(B2M)55Hpl Tg(HLA-B*0702)#Chmb/TdilJ
027849 NOD.Cg-B2mtm1Unc Tg(B2M)55Hpl Tg(HLA-A*1101/H2-Kb)#Sette/TdilJ
12 strains use this protocol