Protocol 31255 - Tyrp1<b> Probe

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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= 149 bp

Wild Type = 149 bp

Ref Seq: GTGCAATGATAATTTCTCAGGACACAACT(G/A)TGGGACTTGCCGTCCTGGGTGGA

Mut = A

WT= G

>chr4:80835311+80835459 149bp CCCCACGATGGTAAAGATGA CCTGTGAGAATTTTCTGGTTGC

Sequence

Wt Sequence:

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction
29465		CCC CAC GAT GGT AAA GAT GA		Forward	A
29466		CCT GTG AGA ATT TTC TGG TTG C		Reverse	A
36484	Fluorophore-1	ACA CAA CTG TGG GAC TTG CC	Quencher-1	WT Probe
36485	Fluorophore-2	AGG ACA CAA CTA TGG GAC TTG C	Quencher-2	MUT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
29465	0.40 uM
29466	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
000571	B6.Cg-Whrn^wi Tyrp1^b/+ +/J
000027	B6.D-Tyrp1^b Dock7^m/J
001045	SI/Col Tyrp1^b Dnah11^iv/J
000064	STOCK a Tyrp1^b Pmel^si/J
002238	STOCK a Tyrp1^b shmy/J
001432	STOCK a/a Tyrp1^b Ndc1^sks/Tyrp1^b +/J
002142	STOCK 11R30m/J
000594	STOCK T(2;8)26H a/T(2;8)26H a Tyrp1⁺/Tyrp1^b/J
001101	STOCK T(3;4)5Rk Tyrp1^b/J
9 strains use this protocol