Protocol 18500: End Point Analysis Assay - H2-Ab1<bm12> EP

B6(C)-H2-Ab1^bm12/KhEgJ

Stock No: 001162

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 =(t)TCCTGGAGC(a)AA(a)
Heterozygote = (a/t)TCCTGGAGC(g/a)AA(c/a)
Wild type = (a)TCCTGGAGC(g)AA(c)

Sequence

SEQ

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
27153		AGA CGC CGA GTA CTG GAA CA		Forward	A
27154		GGC CCC TCG TAG TTG TGT CT		Reverse	A
27155	Fluorophore-1	AGA TCC TGG AGC GAA CGC	Quencher-1	WT Probe	A
27156	Fluorophore-2	AGT TCC TGG AGC AAA AGC	Quencher-2	MUT Probe	A

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
27153	0.40 uM
27154	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	40.0	--	Forever

JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.