Protocol 14434: Pyrosequencing Assay - Hc<0>

B10.D2-Hc⁰ H2^d H2-T18^c/oSnJ

Stock No: 000461

Version 2.0

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

WT = TA/TA

HET= --/TA

Mut = --/--

Sequence

ACAACAACTGGAACTGCA[ta]CTTTGAAATTAAAGAATA

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
11590		ATT TTG CTT CCA CAG GTA TGG T
11591	Fluorophore	TGA CAG GGC AAG TTA GAA CTA AGC
11592		ACA ACA ACT GGA ACT GC

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.00
MgCl2	2.00
dNTPS-kapa	0.20
11590	0.50
11591	0.50
Glycerol	5.00
Kapa 2G HS taq polym	0.01
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
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.

Strains Using This Protocol

Stock Number	Strain Name
003147	B10.Cg-Hc^1 H2^d H2-T18^c Tg(DO11.10)10Dlo/J
000461	B10.D2-Hc^0 H2^d H2-T18^c/oSnJ
000463	B10.D2-Hc^1 H2^d H2-T18^c/nSnJ
034608	J:ARC(S)
004306	NOD.CBALs-Hc^1/LtJ
030511	NOD.Cg-Hc^1 Prkdc^scid Il2rg^tm1Wjl/SzJ
6 strains use this protocol