Protocol 34353: Probe Assay - Tmem131<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

>chr1:36889478-36889588 111bp GCATTTCAGAGTTGCTAAAGTTCTG TGAGGTCAAGACTGGCATCC

Mut= 110 bp

Wt= 111 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CAGGCGTAGATAGGTATTTTCAAGGCACCTTGCACAGCTGGGTTATTGAAGCTGTCTTAGTATTGATCATGACTACATAGCAAAGCATTTCAGAGTTGCTAAAGTTCTGAAGCATTCGCTGTATTTTCCTCGtaaccattactctcatacaagcagtgtttccttggcttgttctggatgccagtcttgacctcagggccctcacaaatgctaattactctacctaccattggtctacattccccagtaagcaatatattaaaatgtaaaattgtatttactgcaaggcaaacggtgaaattgggtttctgttcctatcagtgatgtttggtttgttttcttgacagcattcattcagtcggagagcataatagaagtcctgcgctttgacgacggagggctactacaggtaattctcttgagtaatatgactattcttctttcctgccttggaaaccagtgtaactggttttcctgaagtatgttttatgaactgtagacagtcagtggagttattgcatgaacctcctactagaattactttttagagtaaacatttttctaaacagTGGACTCCCTGTGTAGTATGGAAGCTGTAGGTAAGTAAGTCAGAGTTTGTGTCCTGTGGCTGTGAGGGTATGTAAAAAAAAAAAAAGCCAGGTCAAATGAGGTTGGGTCTGTGAGCTCTCTGGTTGTTAGTGCTGAG

This mutation is a 437 bp deletion beginning at Chromosome 1 position 36,889,104 bp and ending after 36,889,540 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43386 GCA TTT CAG AGT TGC TAA AGT TCT G Common A
43387 TGA GGT CAA GAC TGG CAT CC Wild type Reverse A
43388 ACA GCC ACA GGA CAC AAA CTC Mutant Reverse A
43389 Fluorophore-1 ACA AGC AGT GTT TCC TTG GC Quencher-1 WT Probe
43390 Fluorophore-2 CCT CGT GGA CTC CCT GTG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43386 0.40 uM
43387 0.40 uM
43388 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.