Protocol 36387: Probe Assay - Heca<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

>chr10:17915993-17916143 151bp GGTGTTTGTTCTAGGGGAGGT CTCCCAAAGCTGCAGATCA

Mut = 131 bp

Wt = 151 bp

Fam = Mut

Hex = Wt

Sequence

MUT sequence:

ggtgtttgttctaggggaggtgtaactcgcacgaatgggccaggcggacagatggacgcccTCttttgtcaccaagttaacttagagaattaaaggctgctttgtgggtacttggcgtgtgaggtatgagg

This mutation is a 8090 bp deletion beginning at Chromosome 10 position 17,907,992 bp and ending after 17,916,081 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48611 GGT GTT TGT TCT AGG GGA GGT Common A
48612 CTC CCA AAG CTG CAG ATC A Wild type Reverse A
48613 CCT CAT ACC TCA CAC GCC AAG Mutant Reverse A
48614 Fluorophore-1 CTG AAT CTC GTT TTC TTC CTC TTT C Quencher-1 WT Probe
48615 Fluorophore-2 CCC TCT TTT GTC ACC AAG TTA ACT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48611 0.40 uM
48612 0.40 uM
48613 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.