Protocol 31480: Probe Assay - Pkmyt1<em1(IMPC)J> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

Mutant=  138 bp

Wild Type = 130 bp

>chr17:23733540+23733669 130bp AAAGCCTGGACACAGCAGAG ACAACGGTGCATCAGAGACC

Sequence

Wt Sequence: AAAGCCTGGACACAGCAGAGaCACTGTCCGTGGTTTGGGGGACTACCATTTCCTCTTAGCACTGGACCCTGGTCATAGCACATAGGTCTACCCCACCTCCTCTGTTGCTGGGTCTCTGATGCACCGTTGT

 

Mutant Sequence: AAAGCCTGGACACAGCAGAGacATCTGGGACTACAGCCAGACCCAGGGTTTTGAGACCATACTGGCTACTACTACCCTGCCTCAGTGTATTCTGTTCATCTCTACAGAATGATGATGCCAACCCTAGTAACCATGGAA

1036 bp deletion beginning at Chromosome 17 positive strand position 23,733,561 bp and ending after 23,734,596 bp (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36971 AAA GCC TGG ACA CAG CAG AG Common A
36972 TTC CAT GGT TAC TAG GGT TGG Mutant Reverse A
36973 ACA ACG GTG CAT CAG AGA CC Wild type Reverse A
36974 Fluorophore-1 AGA CCC AGG GTT TTG AGA CC Quencher-1 MUT Probe
36975 Fluorophore-2 CTT AGC ACT GGA CCC TGG TC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36971 0.40 uM
36972 0.40 uM
36973 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.