Protocol 36497: Probe Assay - Tmem67<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

>chr4:12077432-12077550 119bp TGCCTTTCAAGCAAATGATG TCCACTGTGCAAGATATAAAGGAGT

Mut = 138 bp

Wt = 119 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 ACTTCAGTGAATGGTGTTATGTACTGGTACATGTACGTTTATAGGTTTGGTGTCTAAAAATAAAATTTAGTGTTGTGTGGGGAATATGGATTTGGAGGCTATTAGAAGCCCTCACAATTTTGACAACCTGCCAGCAATCTGTGAAGACACTCTCCTCATCCCAGCATGTAAGGAAAGATGTTGAGAAATTTGGCttagggaaaatgttggtttgctgttatagtcacttagaaaactagttctatgtgttttcagttggaatggagaagattttttttggtatattttattatatatatgcttggcatgcctttcaagcaaatgatgctactttgagaattattattacactgataattacagggacatgtgaatatttcagagtatataatacaacactcactcctttatatcttgcacagtggaaagaaatgttagtgggtctttgttagcccaagcaacttgcgagctttgtgatgaaagtgaaaactcatttacaaaagccaatgctttaggaaccaggtgagtagtgtactgaaaacaaaattcattgatgtcctacttttatgtgccatctgtgtatcccaagaaaaaatacaggtagtcattaaaggggcTGAGATTCAGAATCCATGAAAACGTGGGCTCAAGTCTTAAATTAGCTTTCTAATAATTTGGTGTTAGATAGTTAGAATCTCCTGTATTTAGAACGGAAGTACACTCTCCATTGCATAGGTTTCATAAGCTTCT

This mutation is a 423 bp deletion beginning at Chromosome 4 position 12,077,241 bp and ending after 12,077,663 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48910 TGC CTT TCA AGC AAA TGA TG Wild type Forward A
48911 TCC ACT GTG CAA GAT ATA AAG GAG T Wild type Reverse A
48912 AGC ATG TAA GGA AAG ATG TTG AG Mutant Forward A
48913 GAG TGT ACT TCC GTT CTA AAT ACA GG Mutant Reverse A
48914 Fluorophore-1 CAC TGA TAA TTA CAG GGA CAT GTG A Quencher-1 WT Probe
48915 Fluorophore-2 TTG GCT GAG ATT CAG AAT CCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48910 0.40 uM
48911 0.40 uM
48912 0.40 uM
48913 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.