Protocol 37125: Probe Assay - Scoc<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

>chr8:83434239-83434381 143bp CAGGGCAGTCTTTAGTAGCACAG CCATGACAGAACAATCTATGCAG

Mut = 164 bp

Wt = 143 bp

Fam = Mut

Hex = Wt

 

Sequence

MUT Sequence (junction in uppercase):

taagtgcagcatttaacgtcagagtcttgattactgagcagctcagcagcatctgtccttatgcttctctctggaactagttgtcctttgctgatgtatgcatacacacttaatacttcCTtgttcccaggctagagggctctgctgcatagattgttctgtcatggcttctgcatcatcagttacactattcattctatttacaggaaagagtttgtgagtgcccagaatc

This mutation is a 3865 bp deletion beginning at Chromosome 8 position 83,434,286 bp and ending after 83,438,150 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50585 CCA TGA CAG AAC AAT CTA TGC AG Common A
50586 CAG GGC AGT CTT TAG TAG CAC AG Wild type Forward A
50587 GTG CAG CAT TTA ACG TCA GAG T Mutant Forward A
50588 Fluorophore-1 ACC CTT TAT TGA AGT TCT AGG GAA GG Quencher-1 WT Probe
50589 Fluorophore-2 TGC ATA CAC ACT TAA TAC TTC CTT GTT CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50585 0.40 uM
50586 0.40 uM
50587 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.