Protocol 37260: Probe Assay - Tmem144<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

>chr3:79834117-79834216 100bp CAACACCATCAGGGATTTGG GAATGCATCCTCTAATACTCACTG

Mut = 91 bp

Wt = 100 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

CTAGTGACAACATGCAACACCATCAGGGATTTGGCCCTGGGAACCCTCAAGagcgtaaggcaatggttgtgagaatcttagaaggaaagccagtgagtattagaggatgcattctagagttgtgttgaggcagaagttaaattcaggaaaaatggtaatttaactaagaaatgcgtttgttgtgtgggaagtcatccatttttccttcatgttgtatctacaggcaacattgctgtcgttccaattatcaaaaccatcggtttaggacttggaatcctaatctggggatcatttaacaccttaactggctgggcaagctcaaggtaacacagtccacactatctcagctacgagtcctgcatcctgcatttggtgtaaactgggatctagctatattgggagaaataacatgattcttggtctcagagtatggctggtacatgtaaatgaaccatgtctattatttgtctgaagctcttcataagtgatggagatattttatgaagatatagtaaaggaaataaaaagtaaaagctgtatctgggagacaagTGGGGAGAAAGGCAGCTCATGTCTGCTTTCTCCCTCTACTGCCTAACCAGGTACCTAAAGTAGATGCTCGACAATCTGTGGCTCGATCTCCCTGGGATTTTGTAAGATTTAGTGAGATGTAGCTATGTCAAAACACT

This mutation is a 501 bp deletion beginning at Chromosome 3 position 79,833,679 bp and ending after 79,834,179 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50927 CAA CAC CAT CAG GGA TTT GG Common A
50928 GAA TGC ATC CTC TAA TAC TCA CTG Wild type Reverse A
50929 GTA CCT GGT TAG GCA GTA GAG G Mutant Reverse A
50930 Fluorophore-1 AGG CAA TGG TTG TGA GAA TCT TAG Quencher-1 WT Probe
50931 Fluorophore-2 CAA GTG GGG AGA AAG GCA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50927 0.40 uM
50928 0.40 uM
50929 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.