Protocol 37140: Probe Assay - Cchcr1<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

>chr17:35520687+35520798 112bp TTCTTCTTCGCCTGATCTCAC TTCATCTACCACACCACTGTCA

Mut = 109 bp

Wt = 112 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GAAGCCCTCCGATGTGCTTGTTTGACGTTTAACCCATCTGCCTGCTTATGTGTGGATCTCTGGAGAGATGGTGTTCTTCTTCGCCTGATCTCACTAGCCCTtaggtcgactgagcccagaaagtggatgaccacgtgtgcttagtagggacttggtggttgattgacagtggtgtggtagatgaatatatcctcctgcctcagctcagccccttgcttccaccctcccagcagctcaccttttcccctcctgtctttgaaggtctctggagctggggttctccagtgccctgagccagcaggccgagctgatctcacggcagctccaagagttgcggcggctggaggaggaagtccggtccctacgggagacctcgctgcagcagaagatgaggttggagactcaggccgtggagctggatgctctggcagtggcagagaaggccggccaagctgaggccgagggcctgcgcaccgccttggccggagctgaaatggttcggaagaacctggaagaggcgaagcacaaggagctggaggagattcagagcctgcaccaagagcaggtgaacacgggggtgggaggggcttagaagcatatcagggcacgccgtccagTAAATTGCAAGGAGCCTCGGGAGCAGCAGACCCGTGGAGGTGGGGGAAGCCCCCCAAAATACTTGGGTATTTTGGTTTCCCTCAGGCTGCACCTGTTACTTGGGAGTCAGTGAGCTCGTAACCTTCAATA

This mutation is a 516 bp deletion beginning at Chromosome 17 position 35,520,715 bp and ending after 35,521,230 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50647 TTC TTC TTC GCC TGA TCT CAC Common A
50648 TTC ATC TAC CAC ACC ACT GTC A Wild type Reverse A
50649 GGG AAA CCA AAA TAC CCA AG Mutant Reverse A
50650 Fluorophore-1 CCA GAA AGT GGA TGA CCA CGT Quencher-1 WT Probe
50651 Fluorophore-2 CCC TTA AAT TGC AAG GAG CCT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50647 0.40 uM
50648 0.40 uM
50649 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.