Protocol 37791: Probe Assay - Ensa<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

>chr3:95624861+95624963 103bp TGCCTGTCGCTCTAAAGAATC CCTCCTATGAGAGCCAATGAG

Mut = 99 bp

Wt = 103 bp

Fam = Mut

Hex = Wt

 

Sequence

MUT Sequence (junction in uppercase):

tctctttcccggttggtgttctagcttgcctgtcgctctaaagaatccgcccacctccgGAGAAAGCAGGagggagcaggaatggtggacacagatgactggtggcctttgctggcttctgcctgtcctttatagctaattc

This mutation is a 7358 bp deletion beginning at Chromosome 3 position 95,624,895 bp and ending after 95,632,252 bp (GRCm38/mm10). There is a 9 bp AGAAAGCAG insertion at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52157 TGC CTG TCG CTC TAA AGA ATC Common A
52158 CCT CCT ATG AGA GCC AAT GAG Wild type Reverse A
52159 CAG GCA GAA GCC AGC AAA G Mutant Reverse A
52160 Fluorophore-1 ATT GGT GTG TCG TTA CAT CAT TGC Quencher-1 WT Probe
52161 Fluorophore-2 AGA AAG CAG GAG GGA GCA GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52157 0.40 uM
52158 0.40 uM
52159 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.