Protocol 17673 - H2-T18<a/b>-EP-Alternate 2

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

H2-T18<a> = C (Jrs 469, 4804, 465) = "hom"

H2-T18<b>= T (Jrs 666, 664, 457, 438) = "wt"

Sequence

Tctgatgagttattgatctagctatgataa(T/C)cagtgattg(ATTG/----)ctattat(A/C)acagtcaca

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction
25550	Fluorophore-1	AAT CAG TGA TTG ATT GCT ATT ATA AC	Quencher-1	WT Probe	A
25551	Fluorophore-2	TAA CCA GTG ATT GCT ATT ATC ACA G	Quencher-2	MUT Probe	A
25833		AGT CCA AAG TTT TCA CAT TAA TCA		Forward	A
25834		TCA ATG AGA AGC CCT TTT AAG TC		Reverse	A

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
25550	0.40 uM
25551	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

Stock Number	Strain Name
000469	B10.A-H2^a H2-T18^a/SgSnJ
000465	B10.BR-H2^k2 H2-T18^a/SgSnJ
004804	B10.BR-H2^k2 H2-T18^a/SgSnJJrep
036602	B10.Cg-H2^k2 H2-T18^a Tg(SV40-HA)HA104Ajca/VradMmjax
037369	STOCK H2^u H2-T18^a Tg(CD2-TcraAc1-9,-TcrbAc1-9)4Kio/WdcJ
037372	STOCK Il10^tm1Flv H2^u H2-T18^a Tg(CD2-TcraAc1-9,-TcrbAc1-9)4Kio/WdcJ
037371	STOCK Ptprc^a Pepc^b H2^u H2-T18^a Tg(CD2-TcraAc1-9,-TcrbAc1-9)4Kio/WdcJ
7 strains use this protocol