Protocol 33864 - Mvd<em1(IMPC)J>

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

>chr8:122440060-122440191 132bp CCAGGACCAGGTGATTGTG TGTGTGTGGATGAGGAGACA

Mut= 134 bp

Wt= 132 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

CGGTGCTCTTAACCACTGAGCCATTGCTCCAGCCCTTTCTGGGAGATTTGACTGTGCTTGGGGTCGCGGGAGTCCTGGGCAGAGGGGCCGTTCTGATCGGTACCAtctgggacacggtcaggacccggactcctctgggcggacaagcaggtggaggagatagaccaccagccacataggcacatgattcctggtaaacgccctcggctgtatctgggcagtgagagaaagaaggaggaagtgtgaattagaagtggactggtcccctacctccactgtgctctgggaaggttcactgagctttgagtctgctgccaatttaggaggtcttcctgaggttgtgtagtgagaggggatggagcactgaggggaggaccaggaactcactgcttcggttgttgtttcaggggggaagcgagatgaagcgctgatcctgcccatcaactcctccctgagcgtcacgctgcaccaggaccaggtgattgtgctctggtggctggaatggggtgacttctcatgggaccatagccgggCCTGTGTCCTCCTAaTTCCCGTTGACCATCCAATACGCGTTAACCCTTGTCTCCTCATCCACACACAGACCcaaagccacggggttgGTTTTACGTCCGATTCCTTTACTCAAGAGGGACCAGGTGTCCATGGTTCTGTGGTGAGACAGTGCTCACCCTGACACAGCGCAGGTCAGGCCAGTATGGCTACCC

This mutation is a 428 bp deletion beginning at Chromosome 8 position 122,440,127 bp and ending after 122,440,554 bp (GRCm38/mm10). In addition, there are 2 smaller intronic deletions before the 428 bp deletion, neither of which should alter the results of the exon deletion: a 16 bp deletion (CAACCCCGTGGCTTTG) and a single bp (T) deletion that are 71 bp and 15 bp before the 428 bp deletion, respectively.

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction
42360		CCA GGA CCA GGT GAT TGT G		Wild type Forward	A
42361		TGT GTG TGG ATG AGG AGA CA		Common	A
42362		TCT GGG AGA TTT GAC TGT GCT		Mutant Forward	A
42363	Fluorophore-1	ACT TCT CAT GGG ACC ATA GCC	Quencher-1	WT Probe
42364	Fluorophore-2	CCA CCT GTG TCC TCC TAT TCC	Quencher-2	MUT Probe

Reaction A

Component	Final Concentration
Kapa Probe Fast QPCR	1.00 X
ddH2O
42360	0.40 uM
42361	0.40 uM
42362	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.