Protocol 35464: Probe Assay - Stac<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

>chr9:111635117-111635238 122bp CCCATTTCAGGTATCCACCA TGATGGGTCCTGTGACTCCT

Mut= 125 bp

Wt= 122 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GGTGGGGCCTAAGAAAGCAAGGCTGGGAAGGAAGATGGTGGAATGGGTCATGTGGAGCCTCTCCCTGCTCACTAGCTTATGGGATACAGGATGAACTACTGAGTCCCATTTCAGGTATCCACCAGCCTTGGACTGCCCGCCCTGGGGCCTGGCAGAACTTCCCAGgtatacatggggtctagggcagagctggctgcatcccccacaggagtcacaggacccatcataagagaggctgtcctgtcattgccttgcagctccagaaactaaaacggtcactttctttcaagaccaagagcttgcggagcaaaagtgctgacaacttcttcccaagaaccaacagtgatgtgaaaccgcaagcagacctgctggccaaggccagcccaggccccagcccaatagccatccctggaagcccagcgtccatgcccaccaaggctggcctgcaccccggcagcaacagcaagctccacgcctttcaggagcatgtctttaagaagcccaccttctgtgatgtctgcaaccacatgatcgtgggtaagacccctacccccacccctacaggtacaatgtccttgggaaaaggaatgagagctggaggcccaggttcagGGGACCCATAGACCAGCTCTGAGCTTCAGAGAGTTAAATGTAAGAAGGTCAATGTGGGGAGTGGCCATGATGGAGGGGCCTTACCCAAAGCAAAGGCAAACAGGGCTAGGTGGAGGGAGAATTAGTTTGGAATATGGTTGGAACCAAGACAGCAGCTGGCATCGTCCACCAGAAGCCT

This mutation is a 447 bp deletion beginning at Chromosome 9 position 111,634,731 bp and ending after 111,635,177 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46126 CCC ATT TCA GGT ATC CAC CA Common A
46127 TGA TGG GTC CTG TGA CTC CT Wild type Reverse A
46128 CCA CTC CCC ACA TTG ACC T Mutant Reverse A
46129 Fluorophore-1 CAT GGG GTC TAG GGC AGA G Quencher-1 WT Probe
46130 Fluorophore-2 CTT CCC AGG GGA CCC ATA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46126 0.40 uM
46127 0.40 uM
46128 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.