Protocol 31523: Probe Assay - Ctsj<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 transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

>chr13:61002528-61002658 131bp CAGACATGGGGGTGCTTAG GCACTAGCGTTCTCACTACGG

Mutant=  131 bp

Wild Type = 145 bp

Sequence

Wt Sequence: CTATAAAATTTTCTTATGGACAGAAAAAAATGTGAAGTGTCATTGAAAGCATTAGAATGGATTCTCAGTTCCAGATCTTTCACTTTATTTCACTTCCTGTAAtgacttacaacagtccagacatgggggtgcttagcagtgctgtggttctgattcatactcctcagaaggcaggtgtaactgagtctgttatctctttactgtttaaaggacggaccctgcaggtaccgtagtgagaacgctagtgctaatatcacagattatgtgaacctcccaccgaatgagctttacctatgggttgctgtagcatctattgggcctgtctctgctgccattgatgcttctcatgactctttcaggttctacaatggaggtaagcatatttccttctagatgtgaaaaaaatgaagaattgtgacatcccaatgccatgccatgctatgttcttatctataagattttatgtgagtttgagtttctcgattgccttccctgtgcaatcatactgagtcctatctttaaCTGATATGACCCTCTCATGAAAGTAAGTATATCATATGTTATAAAATTTATGTTTAGTTTATTTTTAAACTATCCC

 Deleted Sequence:

tgacttacaacagtccagacatgggggtgcttagcagtgctgtggttctgattcatactcctcagaaggcaggtgtaactgagtctgttatctctttactgtttaaaggacggaccctgcaggtaccgtagtgagaacgctagtgctaatatcacagattatgtgaacctcccaccgaatgagctttacctatgggttgctgtagcatctattgggcctgtctctgctgccattgatgcttctcatgactctttcaggttctacaatggaggtaagcatatttccttctagatgtgaaaaaaatgaagaattgtgacatcccaatgccatgccatgctatgttcttatctataagattttatgtgagtttgagtttctcgattgccttccctgtgcaatcatactgagtcctatctttaa

420 bp deletion beginning at Chromosome 13 negative strand position 61,002,673 bp and ending after 61,002,254 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37053 GGA TTC TCA GTT CCA GAT CTT TCA Mutant Forward A
37054 AAA ATC TAA AGC ATC CAC CAA A Mutant Reverse A
37055 CAG ACA TGG GGG TGC TTA G Wild type Forward A
37056 GCA CTA GCG TTC TCA CTA CGG Wild type Reverse A
37057 Fluorophore-1 TCC TGT AAC TGA TAT GAC CCT CTC AT Quencher-1 MUT Probe
37061 Fluorophore-2 CTG TGG TTC TGA TTC ATA CTC CTC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37053 0.40 uM
37054 0.40 uM
37055 0.40 uM
37056 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.