Protocol 32804: Probe Assay - S2bpcox16<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

>chr12:81510760-81510896 137bp GCCGTATCAAAGAGGATGG AGGCCAAGATGGAATCAGA

Mut= 124 bp

Wt= 137 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AATATCACCAACAGGATCAAAATCAAGTGTGGGCAGAAGCACTTATATCAAGACTCAACCATACTATTTCCCATGTTATATATATGTACATATATATTTATATTTCATCTAGCCAGTCATTTTGATCACAGTTGTGATCACAGTACCTGTAgtactgcctggttcgaattagcaatctaggtacttttgaactttgttcataatgacttattgactttgcccatgactgtgttttagcttacctcacagtctgaattattagtctctcttccccactagaccatttgcctctcctttcttctcctctaatctttctttcttcttcgtccctctctcctcactcttccatgattttctcagggctgggcttcaacatcgtcggtgggacagatcaacagtatgtctccaacgacagtggcatctacgtcagccgtatcaaagaggatggggctgcggcccaggatgggcggctccaggagggtgataagatcctctcggtgagaaatggcttcggctttctttgctatactttgctccctagaCCACATCTGATTCCATCTTGGCCTGCCTGTTTGGAAATGAAAATATTTAGAGTGTATCATTGAATAGGAAGAACATTGATCAGTCTTCCAACTTTACCGCTGGGATGGGAGGATAGTATTATTCTTGTTTGACAGACAAGGCAAATTTA

This mutation is a 391 bp deletion beginning at Chromosome 12 position 81,510,784 bp and ending after 81,511,174 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40033 GCC GTA TCA AAG AGG ATG G Wild type Forward A
40034 AGG CCA AGA TGG AAT CAG A Common A
40035 GAC TCA ACC ATA CTA TTT CCC ATG T Mutant Forward A
40036 Fluorophore-1 TCG GCT TTC TTT GCT ATA CTT TG Quencher-1 WT Probe
40037 Fluorophore-2 TGT GAT CAC AGT ACC TGT ACC ACA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40033 0.40 uM
40034 0.40 uM
40035 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.