Protocol 37425: Probe Assay - Pycr3<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

>chr15:75919296-75919442 147bp CAACAGAAACGGCAGTGCT GGACAGAAGGACGAGAGCTG

Mut = 153 bp

Wt = 147 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

AACAGAAACGGCAGTGCTATTTAAGTACGATGAATTGACTTGATCAGccccaaggacaagcaaccctaaggagccttaggctcacactgggctgggcaagctgtgtcctatcttcccagtccttaccagctctcgtccttctgtccaggcaaagtagaagctaaacaagtgctggccagtgcaccaacggacaacaacctctgccacttcagggtgagtgggacctagagggccttgtctttctgaagcatttatcaggggcaggtatggcagacggtttgagatggtttggtctgtccaggggaactaggcgtcagaggccaggaagaaagttctggaaggactagcatagatgcagtgtgtgtcttatgtagggtgggtgtggacactcattaggagggaaggttctagaaggcacaacttaagagtttgaggagcagggcctgttgggtttgtcgtaggcgttggagagtacagcaagtacagacattggtgacccACGGGATACGGAATGGTCGATGGCACGCAGGTCAGCCTGTGTAGGATGCTGCCTCACACAGCCTCATTTCTGGCCCTTGTGTCTCTTTAGGCTCTGGGTTGCCAGACTACTCACTCCAACCATGAGGTGCTGCAAAACTGCCCACTTGTCATCTTTGCCACCAAACCCCAAGTCCTGCCAACTGTCCTGGCGGAAGTGGCCCC

This mutation is a 452 bp deletion beginning at Chromosome 15 position 75,918,943 bp and ending after 75,919,394 bp (GRCm38/mm10). There is a 2 bp insertion (AA) at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51315 CAA CAG AAA CGG CAG TGC T Common A
51316 GGA CAG AAG GAC GAG AGC TG Wild type Reverse A
51317 GGC AAC CCA GAG CCT AAA GA Mutant Reverse A
51318 Fluorophore-1 CTG GGC TGG GCA AGC TG Quencher-1 WT Probe
51319 Fluorophore-2 AAA CGG GAT ACG GAA TGG TCG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51315 0.40 uM
51316 0.40 uM
51317 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.