Protocol 33339: Probe Assay - Thsd4<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

>chr9:60428558-60428657 100bp GACCCAGGCTGAATTCTGTG CCTTAGAGGCTTGGGAAAGA

Mut= 122 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

AGCCATCACAGTGCTCAAAGCCAGGGCTGCACTGTGACTCTCCTGGTCACTTAAATTGGTCTCTCATGGCTGAGTCCATATAAACTGAGACCCAGGCTGAATTCTGTGCATTCTGTTTTGCTTCTTAATTGTACCCTGTgtccacatgggtccagtagagaaatgagttctttcccaagcctctaaggttcaagactctaaccagcctctgtttctataggtcccacaacggatggcagtgaccgagggaaccccagaggacagtggcagtggctccccaggtgtgtggggctcctggggcccctggtctgcctgttctcgcagctgcagtggtggtgtgatggagcagacacggccctgcctgcccagctcctaccgtgctcgaggaggctcccggcccaatggccgtgcactgtccatcactggccacgttgtgtcagccgtacggacctcagtaccactgcatcgaagccaggaggaccaaagagccctggctggctccaatgctagccgccaaggccctgcagtggtgcgaggcagccgacatccacaggcccggggccgtgaaccctcagagagaaggtatgtgcacatcttggtccctgtgtctgtccctgcctggacaacagcagagccactaccttgattctgcagaATGGGAGGAATTCTTGCCACCATATTCATATGTGCCCATGGAGGCCAATGGAACCATAGGACAGCATCTGGGTCGATTCTTCAAGCCAAAACCTACTCTTGTTTTATTTAAAAACAAACAAACAAACAAACA

This mutation is a 516 bp deletion beginning at Chromosome 9 position 60,428,091 bp and ending after 60,428,606 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41173 GAC CCA GGC TGA ATT CTG TG Common A
41174 CCT TAG AGG CTT GGG AAA GA Wild type Reverse A
41175 CCA GAT GCT GTC CTA TGG TT Mutant Reverse A
41176 Fluorophore-1 TCC ACA TGG GTC CAG TAG AGA Quencher-1 WT Probe
41177 Fluorophore-2 TGT ACC CTG TAT GGG AGG AAT T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41173 0.40 uM
41174 0.40 uM
41175 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.