Stock No: 000465
Protocol 21974: End Point Analysis Assay - H2<k>rs8249979 Alternate 1-EP
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

Sequence

Mut = T H2<k> Jrs:  646, 656, 465, 4804

WT= C other haplotypes

tcccaccaTGAGCCTCTACCCTCTAGGAATTCG(c/t)CAGGGACCAGTCAGGTC

ACTCAGCTggggcttct

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
21918 Fluorophore-1 TAG GAA TTC GTC AGG GAC CAG Quencher-1 MUT Probe
21919 Fluorophore-2 AGG AAT TCG CCA GGG ACC Quencher-2 WT Probe
22189 ATC CCA CCA TGA GCC TCT AC Forward A
22190 CAG CTG AGT GAC CTG ACT GG Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
22189 0.40 uM
22190 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 4.0 -- Forever
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
000465 B10.BR-H2k2 H2-T18a/SgSnJ
004804 B10.BR-H2k2 H2-T18a/SgSnJJrep
036602 B10.Cg-H2k2 H2-T18a Tg(SV40-HA)HA104Ajca/VradMmjax
000666 C57BL/10SnJ
000486 MRL/MpJ
026243 NOD.Cg-(D9rs4135590-D9rs13480186)H2k2Tg(ILK3mHEL)3Ccg Tg(TcrHEL3A9)1Mmd/SlsgJ
6 strains use this protocol