Protocol 45383: Probe Assay - Myh15<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

>chr16:49069147+49069236 90bp AGCAGTTAGTTCATCTCCCAGAG GTGATTTCTAACAGAAGCCAACAG

Mutant=  123 bp

Wild Type = 90 bp

Sequence

Wt Sequence: agcagttagttcatctcccagagatcaagaggcaaTacgaggtcttgatgtcagcttaacccagatctgttggcttctgttagaaatcac

Mut Sequence: agcagttagttcatctcccagagatcaagaggcaaTTcatttaaagagactccttatgaactgtattgtacaagctagttaattccttctcagggaagagcccttcagtcttgcgaatagtca

 

A 1220 bp deletion beginning at Chromosome 16 position 49,069,183 bp and ending after 49,070,402 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65476 AGC AGT TAG TTC ATC TCC CAG AG Common A
65477 GTG ATT TCT AAC AGA AGC CAA CAG Wild type Reverse A
65478 TGA CTA TTC GCA AGA CTG AAG G Mutant Reverse A
65479 Fluorophore-1 AGA GGC AAT TCA TTT AAA GAG ACT CC Quencher-1 MUT Probe
65480 Fluorophore-2 CGA GGT CTT GAT GTC AGC TTA AC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65476 0.40 uM
65477 0.40 uM
65478 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.