Protocol 46059 - Ttc12<em1(IMPC)J> Alt 1

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

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

>chr9:49470370-49470462 93bp TAACGGCAGCGAAAAATAAG GCACAGAGTCAGCCCTCAGT

Mutant= 87 bp

Wild Type = 93 bp

Sequence

Wt Sequence: taacggcagcgaaaaataagcagcgctgggccaaactaGaaaaccggcgatctagaaggaccttccgacgtacactgagggctgactctgtgc

Mut Sequence:taacggcagcgaaaaataagcagcgctgggccaaactaGAGTGATggcaaatgtttaccaacaggctgcaagtgccagtttccatgg

A 321 bp deletion beginning at Chromosome 9 position 49,470,103 bp and ending after 49,470,423 bp (GRCm38/mm10). There is a 5 bp insertion ( AGTGA ) at the deletion site.

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction
66144		TAA CGG CAG CGA AAA ATA AG		Common	A
66145		GCA CAG AGT CAG CCC TCA GT		Wild type Reverse	A
66146		CCA TGG AAA CTG GCA CTT G		Mutant Reverse	A
66147	Fluorophore-1	CCG GCG ATC TAG AAG GAC C	Quencher-1	WT Probe
67120	Fluorophore-2	ACT AGA GTG ATG GCA AAT GTT TAC CA	Quencher-2	MUT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
66144	0.40 uM
66145	0.40 uM
66146	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.