Protocol 37491: Probe Assay - Tmem179<em2(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

>chr12:112504549-112504656 108bp CTTCCTGAACCTCCTGGTCA CCCTTCTCAGTGATGGTGTCA

Mut = 102 bp

Wt = 108 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

TGAGTTCTTGACTTGGCCCTGGTTCCCCGGTACATCAGTGGTGGCAAGTCCCCAACTCCCAGTCTGCGAGAGAACAAGATGGCAGCTCAACATCGGAACatttgcagggatggcagcagaggagatggaagcagccctctgagccccattccatcccatcagggccccaggtgctcaccagaaaacctggacatcccccctcctgtttcaaggggagctatttggggtcagaccttgagtggctccttcagcatccatctcttgactccactgcagctccttcttctacgccttcctgaacctcctggtcagtgcctttgtggtcttcctggtcttcattgccagcaccatcgtgagtgtgggcttcaccatgtggtgtgacaccatcactgagaagggctccacaccccacaggtatgcctcacccctgcttcatcagccacctacaagacccacctttacctgggtccctatgtttttcttcctgaatctgaggctttgcccacctgtgccccacccctacacctgttgccttttccctaagacccacccacctgcgccccacccccgcacctgttgccatgcccctaagacccgcccacctgcgccccccccacacctgttgccatgcccctaagacccgcccactcaatgtcaggcacctctttggtctagtagccctacagcattacacatggaaggtccctgtgggccgagaggcaccagatgggatcctgcagttgacaccgcctggcacacttgctagccttagctcgctctgaagctaactccactgtgtcgccatgatgttagtttgtggttggtgagtggagattgccctcatggtgtccctgtgacacttatgcagggacacagtgcaatgtgcctcagggggcatcacatccatctcagactgggaactgcaggactgactagacagagcagctcatgaccccgagtgccggacagacagtgacaagatagtgctccgggccagtcgtaaaggtctgcagctcctcttctttgaagcctgtcttgctccacccagtgaggggccaaagctgaccccgagtggagttcctagaggcatctgtccctttgggtactggcccctctgtcttagggttttgactgctgcaatggaacaccccggccaacgcgtcttggggaggaaagggtttattcagcttacacttcaatatcacagttcctcatcaaagaaagtcaagacatgaacacagcagggcagggacctggaggcaggacctgaggcagaagccactggaggggtgctgcttactggcttgctcctctagatctgctcagccaggggtggccacgcccacaatgggtggggctatttcccaccaatcactaagtaagaaaatgtcttacaggcttgtctgcagcctggaagcattttctcaattgaggttcctgcctctcgactttagcttgtatcaggttgatctaagacgccactcagtggttaggggaagtattctctgcatcctagctctgctgccttttgacttgggggctcaggggctcggggcctggggggctcgggggcttgggggctcaggggctcacaagtcaccagccacttggcttcgtgctattttagctgcgaggagttccaggaaacggacttggagctgaacgtcgacaactctgccttctaccatcagtttgctatcgcccaggtaggcggggtctcgggcaggtaggcggggtctgtctctggccggaagggaatggctctgttggggtgaggggtgagacctcgacagatagaacagactcccggatcccagataaggaaacagactcagatacggtggggccatgCCGGGGAGGTGCAGGCTGTGGAGCAAGAGATTTCCTAGGAGAGGTTAGGAGAGTGGGGTCTCTGAGCAAGACTTGTGTCAGGGGTTGGGAGATAGGGGAGTCAGCAAAGGCAGAC

This mutation is a 1752 bp deletion beginning at Chromosome 12 position 112,503,097 bp and ending after 112,504,848 bp (GRCm38/mm10). There is a 3 bp insertion (CTA) at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48467 AGA GAA CAA GAT GGC AGC TCA Mutant Forward A
48470 TGC TCA GAG ACC CCA CTC TC Mutant Reverse A
51461 CTT CCT GAA CCT CCT GGT CA Wild type Forward A
51462 CCC TTC TCA GTG ATG GTG TCA Wild type Reverse A
51463 Fluorophore-1 CCT GGT CTT CAT TGC CAG C Quencher-1 WT Probe
51464 Fluorophore-2 AAC CTA CCG GGG AGG TGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48467 0.40 uM
48470 0.40 uM
51461 0.40 uM
51462 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.