Protocol 36268: Probe Assay - Mtx2<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

>chr2:74859275+74859410 136bp CTTGCTTATCATTTTTAAGGGGAGT AAATGCCTGTGAAGAAAAAGAG

Mut= 124 bp

Wt= 136 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 ATTAAACCATCTTTAAAAATTTGATATTATTCATTTAAGAAAGTAAAGCAATATGTAATATTGCTTACATACACAAATAGAAAATGTAAAATTTTCCTTAAATTTTAAAATTTTATATATTTACTAATGACTTAAGTCTTTTTTTAAGTAAAATATTTTTGTTTGGAAACTTTAAACTTGCTTATCATTTTTAAGGGGAGTACTACTGTATGAACTTTTAAGTTTAGgaaaagaatgaaggaagagttctctttaaatatgcacaagtttttaataaccttattttatttctctttttcttcacaggcatttttgcagatgtgtaatctgcctgtcaaagtggtgtgtagggcaaatgcggaatatatgtctccatctggtaagtgggtgtgttttctattaatacttttgtttgttatttgacattgaggattgaacctaagaccttagctgtgatagcaggtgcattaccactggccttatttctagcacccaacgttaaagtccttagtttgtttgtttgcttgattttttttctgctttgaaggattatttttctattgtttttttaaagtagcagttatgaacttaatcataaagacctatgtgtacttacttgagctctggtccgagGTGTTTTACTGGATTGCTAAGAACATAAGTGATGTCAGAATTCCTTAGTTTTATTTATAGGGCTTTCCTTACCAGGAAACAAATTTTAATGCTGATGAATCTGGTTTTTGTATGGTTTTGTTTGTTTGTTTTTTTATTTACATTCCAGCCATTGCACCCCTCCCCCCTTCCCTGTCGCCCCTCCCACAGTTCCCCATCCCATTTCTCCTCCACTTGTCTCTGAGAGGGTGCTCCGCTCCCACCAAACCAGCTTCCCTGAGGCTTTGTAGTACAGCTTGAGGTCTGGGATGGTGATTCCCTGG

This mutation is a 406 bp deletion beginning at Chromosome 2 position 74,859,326 bp and ending after 74,859,731 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48230 CTT GCT TAT CAT TTT TAA GGG GAG T Common A
48231 AAA TGC CTG TGA AGA AAA AGA G Wild type Reverse A
48232 GGT AAG GAA AGC CCT ATA AAT AAA AC Mutant Reverse A
48233 Fluorophore-1 AGG AAG AGT TCT CTT TAA ATA TGC ACA A Quencher-1 WT Probe
48234 Fluorophore-2 AAG TTT AGG TGT TTT ACT GGA TTG CTA A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48230 0.40 uM
48231 0.40 uM
48232 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.