Protocol 30765: Probe Assay - Braf<tm1Mmcm> Probe

B6.Cg-Tg(Tyr-cre/ERT2)13Bos Braf^tm1Mmcm Pten^tm1Hwu/BosJ

Stock No: 013590

Version 1.0

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=  84 bp

Wild Type = 132 bp

>chr6:39578176-39578307 132bp ACTGGCTTAGTAGTAGATGAGATTTGA CCAGGCTCTTTATGAGAATACAGT

Sequence

Wt Sequence: gagtatttttgtggcaactgctttagctcatctaactggcttagtagtagatgagatttgaaacctaagaaaattatttaacagacatgaagaaacgatttttctttttgtgtctcAGTCCACGgagctcctttctgacatcactgtattctcataaagagcctgggccgctttcccagcagagttaaagtgt

Mutant Sequence: CAACTGCTTTAGCTCATCTAACTGGCTTAGTAGTAGATGAGATTTGAAACCTAAGAAAATTATTTAACAGACATGAAGAAACGATTTTTCTTTTTGTGTCTCgaatcaacaagtttgtacaaaaaagcaggctccgaattcgagataacttcgtataatgtatgctatacgaagttattctagatgacctgcgagggacgctgtaattgacgacgcaaataaagagaggacctGAGCTCCTTTCTGACATCACTGTATTCTCATAAAGAGCCTGGGC

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
35428		CGA GGG ACG CTG TAA TTG A		Mutant Forward	A
35431		CCA GGC TCT TTA TGA GAA TAC AGT		Common	A
35432		ACT GGC TTA GTA GTA GAT GAG ATT TGA		Wild type Forward	A
35433	Fluorophore-1	CGA CGC AAA TAA AGA GAG GAC CT	Quencher-1	MUT Probe
35434	Fluorophore-2	TGT GTC TCA GTC CAC GGA GCT C	Quencher-2	WT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
35428	0.40 uM
35431	0.40 uM
35432	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
013590	B6.Cg-Tg(Tyr-cre/ERT2)13Bos Braf^tm1Mmcm Pten^tm1Hwu/BosJ
033760	STOCK Braf^tm1Mmcm Gt(ROSA)26Sor^tm3(CAG-EYFP)Hze Nkx3-1^tm4(cre/ERT2)Mms Pten^tm1Hwu/AbshnJ
038467	STOCK Braf^tm2Mmcm/J
3 strains use this protocol