Genotyping Protocols Database
Stock No: 002448
Protocol 18706: End Point Analysis Assay - H2<b>rs253049200-Alt 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

AAGCAGACACAGGAGAGAGGATGTTCTGTaaaacaagcatgtga

Aaggatgcg(A/-)tg(CGTG/-----)atgaaggattctttgctaaaGACACA

ATTATGGATCTGCAA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
21943 Fluorophore-1 ATG CGA TGC GTG ATG AAG Quencher-1 MUT Probe
21944 Fluorophore-2 AAG GAT GCG TGA TGA AGG AT Quencher-2 WT Probe
22205 CAC AGG AGA GAG GAT GTT CTG Forward A
22206 AGG GTT GCA GAT CCA TAA TTG Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
22205 0.40 uM
22206 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 -- repeat steps 2-3 for 40 cycles
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
000438 C3.SW-H2b/SnJ
000664 C57BL/6J
000666 C57BL/10SnJ
001952 C.B10-H2b/LilMcdJ
002448 129S1/SvImJ
002591 NOD.B10Sn-H2b/J
002845 C.B-H2b Tg(H2-Dd)D8Gja/LilJ
7 strains use this protocol