Protocol 45657: Probe Assay - Tgm7<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

>chr2:121106770-121106869 100bp GCAGGTAACACCACCTCCTG CTCACTGGATGGGAAATTGG

Mutant=  109 bp

Wild Type = 100 bp

Sequence

Wt Sequence: GCAGgtaacaccacctcctggagggaggatgggcttgctgggaactgttagctaacaaagggcActgttcgaggtgctggccaatttcccatccagtgag

Mut Sequence:  ttcccaaaagtctggagttatctaataattttcatagtctatattttttatagcatagattttgttgctagCActgttcgaggtgctggccaatttcccatccagtgag

A 417 bp deletion beginning at Chromosome 2 position 121,106,807 bp and ending after 121,107,223 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66154 Fluorophore-1 CTT GCT GGG AAC TGT TAG CTA AC Quencher-1 WT Probe
66155 GCA GGT AAC ACC ACC TCC TG Wild type Forward A
66156 CTC ACT GGA TGG GAA ATT GG Common A
66157 TTC CCA AAA GTC TGG AGT TAT C Mutant Forward A
66158 Fluorophore-2 TGT TGC TAG CAC TGT TCG AGG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66155 0.40 uM
66156 0.40 uM
66157 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.