Protocol 45830: Probe Assay - Mmgt1<em1(IMPC)J>
Version 1.0

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

chrX:56596142-56596270 129bp AATTGTGGCAGCAGATAGCAT AAATGGGAACGTGAAAGGTG

Mutant=  126 bp

Wild Type = 129 bp

Fam = Mut

Hex = Wt

X-LINKED

Sequence

Large deletion:  Mutant sequence with junction in uppercase

taccaggctttcgagttctacgcccacagccatttagcaacgccaccagccccaccttcttctcctcccaggcctttccgtaagtcccgccccttctccaaatccggcgcgtatttgcggacgctctacctcgTCtagtatgatgctgatttttaaatgtgtactgtaaatgatctgtaaaatcttcctttattttttgtttttaacattataatacaattatatagtttctccttttcctctactccctctaatgacttacatgtaccctccttgccatatttcaaattcatggcctctttttc

This mutation is a 12794 bp deletion beginning at Chromosome X position 56,585,378 bp and ending after 56,598,171 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66551 Fluorophore-1 CGG ACG CTC TAC CTC GTC TAG TAT GA Quencher-1 MUT Probe
66552 Fluorophore-2 ATC TCT GCC ATA GTG ACC TTT GAT Quencher-2 WT Probe
66553 AAT TGT GGC AGC AGA TAG CAT Wild type Forward A
66554 AAA TGG GAA CGT GAA AGG TG Wild type Reverse A
66555 CCT CCC AGG CCT TTC CGT A Mutant Forward A
66556 AGG AAG ATT TTA CAG ATC ATT TAC AG Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66553 0.40 uM
66554 0.40 uM
66555 0.40 uM
66556 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.