Protocol 22061: End Point Analysis Assay - Thy1<a>End Point

BKa.Cg-Ptprc^b Bmi1^tm1Ilw Thy1^a/J

Stock No: 017351

Version 4.0

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 = G/G

Heterozygote = A/G

Wild type = A/A

 

Thy1<b> is WT and has A at the indicated location

Thy1<a> is Mut and has G at the indicated location

Sequence

GACCCGAGAGAAGAGGAAGCACGTGCTCTCAGGCACCCTTGGGATACCCGAGCA

CACGTACCGCTCCCGCGTCACCCTCTCCAACCAGCCCTATATCAAGGTCCTTACCC

TAGCCAACTTCACCACCAAGGATGAGGGCGACTACTTTTGTGAGCTTC(a/g)AGTCT

CGGGCGCGAATCCCATGAGCTCCAATAAAAGTATCAGTGTGTATAGAGGTGAGAC

TGGTTCCCAGAAAGATAAAATGTCCAGGTTAGCCAGGCTGGGGTAGCCAATAAAA

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
16073		CTC TCC AAC CAG CCC TAT ATC		Forward	A
16074		GAA CCA GTC TCA CCT CTA TAC AC		Reverse	A
16075	Fluorophore-1	GTG AGC TTC AAG TCT CGG G	Quencher-1	WT Probe
16076	Fluorophore-2	GTG AGC TTC GAG TCT CGG G	Quencher-2	MUT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
16073	0.40 uM
16074	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
001317	B6.Cg-Gpi1^a Thy1^a Igh^a/J
034140	B6.Cg-Il17a/Il17f^tm1.1Impr Thy1^a/J
039952	B6.Cg-Nr1h3^tm1.1Gust Nr1h2^tm1.1Gust/J
038742	B6.Cg-Thy1^a Rbm5^tm1.1Ozg/PkocJ
035728	B6.Cg-Thy1^a Tg(CD2-Tcra,-Tcrb)27Glkm/J
005023	B6.Cg-Thy1^a/Cy Tg(TcraTcrb)8Rest/J
000406	B6.PL-Thy1^a/CyJ
025854	B6;FVB-Ptprc^a Tg(CAG-luc,-GFP)L2G85Chco Thy1^a/J
017351	BKa.Cg-Ptprc^b Bmi1^tm1Ilw Thy1^a/J
007686	BKa.Cg-Sox17^tm2Sjm Ptprc^b Thy1^a/J
036333	C.Cg-Rag2^tm1Fwa Thy1^a/AkhoAbhmJ
036332	C.Cg-Thy1^a Tg(DO11.10)10Dlo/AkhoAbhmJ
005307	CBy.Cg-Thy1^a Tg(TcraCl4,TcrbCl4)1Shrm/ShrmJ
005443	CBy.PL(B6)-Thy1^a/ScrJ
024476	NOD.Cg-Stat4^tm1Gru Thy1^a Ifngr1^tm1Agt Tg(TcraBDC2.5,TcrbBDC2.5)1Doi/LmbrJ
004483	NOD.NON-Thy1^a/1LtJ
005651	SJL.AK-Thy1^a/TseJ
003961	SJL.Cg Thy1^a-Noxo1^hslt/J
025855	STOCK Ptprc^a Lag3^tm1Doi Tg(CAG-luc,-GFP)L2G85Chco Thy1^a/J
19 strains use this protocol