Protocol 43687: Probe Assay - Myo1b<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

>chr1:51834529-51834638 110bp GCTTTGAAGCAAATCACAGAG TCCGACAAAGCAAAGCTACA

Mutant=  82 bp

Wild Type = 110 bp

Sequence

Wt Sequence :  gctttgaagcaaatcacagaggctcttagaAtacccaggggtacccgatgaacatatattaaccagctgaatcctcccacatttttttcttgtagCTTTGCTTTGTCGGA

Large deletion:  gctttgaagcaaatcacagaggctcttagaATctggaaggagatgctgttgcaaggcagcaggaaaggagccaggtagccct

 

A 190 bp deletion beginning at Chromosome 1 position 51,834,418 bp and ending after 51,834,607 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
63700 GCT TTG AAG CAA ATC ACA GAG Common A
63701 TCC GAC AAA GCA AAG CTA CA Wild type Reverse A
63702 AGG GCT ACC TGG CTC CTT T Mutant Reverse A
63703 Fluorophore-1 AAG GAG ATG CTG TTG CAA GG Quencher-1 MUT Probe
63704 Fluorophore-2 ACC AGC TGA ATC CTC CCA C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
63700 0.40 uM
63701 0.40 uM
63702 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.