Protocol 19658: Standard PCR Assay - H2-D1<tm1Bpe> Alternate3
Version 1.0

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 = 257 bp
Heterozygote = 160 bp and 257 bp
Wild type = 160 bp

Two protocols are provided because of different band sizes.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
28995 CTG GTT GCC CCA GCT TC Wild type Forward A
28996 CGA TTT ATA TTC CTC CAG GGT A Common A
28997 GAG GAC ATT CCA ATC ATA GGC Mutant Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
28995 0.50 uM
28996 0.50 uM
28997 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
019995 B6.129P2-H2-K1tm1Bpe H2-D1tm1Bpe/DcrJ
025216 NOD.Cg-Prkdcscid H2-Ab1em1Mvw H2-K1tm1Bpe H2-D1tm1Bpe Il2rgtm1Wjl/SzJ
023848 NOD.Cg-Prkdcscid H2-K1tm1Bpe H2-D1tm1Bpe Il2rgtm1Wjl/SzJ
3 strains use this protocol