Protocol 42295: Probe Assay - Mxd4<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=  123 bp

Wild Type = 123 bp

chr5:34178963-34179085 123bp CTTCAGCCTTGAGGTAAATCC AATCCCACAGAACTAGGCTCA

Sequence

Wt Sequence (deletions in lower case): CAGGCTGCTCCGGAGGGGAAATATTATCTAGGTCAGGAGGGAATTCCTGGAGAGGGATCTAACTACTTCAGCCTTGAGGTAAATCCCCAGGCCCAAAGGCAAGCCTACAGCAGCGcagaggcccatggagggtctggctggccacttcccaccttgcaccagacacatgagcctagttctgtgggatttgctgcctagcttgatagtctatctctggctctttcagacgagctaaactcaggctctacttggagcagctcaagcagctggtacccctgggccctgacagcacacgtcacactactctgagccttctgaagcgtgctaagatgcacatcaaggtaagtctcctcagctaccctgatgccccccagccatccccacccttgGGCAGCATTGGTTCAGAGGACCTGGAGGTGGGTGGTGGAGCCACCTAGCAGGTACGTGTCAGGAGCTTATGGGGAGGCCCAATGGGAGCTG

 

This is a 274 bp deletion beginning at Chromosome 5 position 34,178,762 bp and ending after 34,179,035 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
60338 CTT CAG CCT TGA GGT AAA TCC Common A
60339 AAT CCC ACA GAA CTA GGC TCA Wild type Reverse A
60340 CCC ATA AGC TCC TGA CAC G Mutant Reverse A
60341 Fluorophore-1 AGC AGC GGG CAG CAT TG Quencher-1 MUT Probe
60342 Fluorophore-2 TCT GGC TGG CCA CTT CCC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
60338 0.40 uM
60339 0.40 uM
60340 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.