Protocol 31546: Probe Assay - Hspb9<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

Mutant=  117 bp

Wild Type = 137 bp

>chr11:100713810+100713946 137bp GGAAGATAAGACAGCGACTTGA GCATCCGAGAGTGCAACTG

Sequence

Wt Sequence (deleted region lower case): GATGGAGCTGTAACCCCGCACATTCAGGTGATCCAGTTCCTTCATAGCCTCCTTCCTGCCCTCTGATGTGAGAGAAGGGCCCAGGGAGAAGCAGAAGCCGGTTCTGGACAAAAGCTGCAGCGCTTGCAAGGAAGATAAGACAGCGACTTGAGTGTTTTTGCCCTTCTGGATGGGAAGAGTGGgcgggcccttcctcttcgcacccgccccggatggggggttggggagccctcgcgccctcaggcagcagttgcactctcggatgcagcgggtaggaagcagtttctccaccgggcagcgcgaacccggggaaaaccgggtagcttcccgatgtcccagcgtggcccttgctgaacggaatcaagtggccactctaccagtgaggctgctcagggatgaggtgcagggcaatggctgtgagcaaccaagtttccaaataaaggtggatgcccaaggtttcgctccggaggacttggtgGTGCGGATAGACGGCCAGAATCTGACGGTGACCGGCCAGCGGCAACACGAGTCGAATGACCCGAGCAGGGGCCGCTACCGCATGGAGCAGAGTGTTCACCGACAAATGCAGCTCCCGCCGACCTTAGATCCTGCAGCCATGACCTGCAGCCTGACCCCCTCTGGCCATCTGTGGCTCAGGGGACAAAACAAGTGCCTACC

 

296 bp deletion beginning at Chromosome 11 positive strand position 100,713,863 bp and ending after 100,714,158 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37116 GGA AGA TAA GAC AGC GAC TTG A Common A
37117 TCG GGT CAT TCG ACT CGT Mutant Reverse A
37118 GCA TCC GAG AGT GCA ACT G Wild type Reverse A
37119 Fluorophore-1 TCT GAC GGT GAC CGG C Quencher-1 MUT Probe
37120 Fluorophore-2 CCC TTC CTC TTC GCA CC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37116 0.40 uM
37117 0.40 uM
37118 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.