Protocol 46662: Probe Assay - Tex47<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

>chr5:7304772+7304896 125bp TCACTACCATTGAGACTTGTAATCA GAACCTTGGGCAGAAGAAGA

Mutant=  98 bp

Wild Type = 125 bp

Sequence

Wt Sequence: TCACTACCATTGAGACTTGTAATCATCTAAAAAAGAAATAAGATTCATCATGTCTTTCATGGTcCATAATCGGAAGGGCAGCAAAAAGCAATTTCAAGTGGATCCTCTTCTTCTGCCCAAGGTTC

Mut Sequence: TCACTACCATTGAGACTTGTAATCATCTAAAAAAGAAATAAGATTCATCATGTCTTTCATGGTccGATATTGTGTGGCCAGCTCCTTCCCGTTTCTAG

A 713 bp deletion beginning at Chromosome 5 position 7,304,836 bp and ending after 7,305,548 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
68561 TCA CTA CCA TTG AGA CTT GTA ATC A Common A
68562 GAA CCT TGG GCA GAA GAA GA Wild type Reverse A
68563 CTA GAA ACG GGA AGG AGC TG Mutant Reverse A
68564 Fluorophore-1 TGT CTT TCA TGG TCC GAT ATT GTG T Quencher-1 MUT Probe
68565 Fluorophore-2 AAG GGC AGC AAA AAG CAA TTT C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
68561 0.40 uM
68562 0.40 uM
68563 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.