Protocol 46358: Probe Assay - Map10<em1(IMPC)J> Alt 2
Version 3.0

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

>chr8:126397180-126397328 149bp AGAAGCCATGTCCCCAGA TCTCAAATGCACGCCTTC

Mutant=  152 bp

Wild Type = 149 bp

Sequence

Wt Sequence: agaagccatgtccccagagctagccttactgtgtaagaccgcttcttcccaggacttctggacaactgaggcgtctgagtcccctgcagaccagctgttggtatccttggcacatggctctgagccctccagaaggcgtgcatttgaga

Mut Sequence:  CACTGTGTATCCTGGAAGTTTATTTATTACTAACTCGCAGATGTCTTTGccGTGGTCGCCATTGCTGTTGCCCCGGGCGACGCCGCCAGCTCGCTCCGCCAACCGGAAGCGGGAgcgagggtcacgtgaccggccgcgccctcgcgagacttgacttccggc

A 2611 bp deletion beginning at Chromosome 8 position 125,669,882 bp and ending after 125,672,492 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
67619 GCC GGA AGT CAA GTC TCG Mutant Reverse A
67620 Fluorophore-1 AGA TGT CTT TGC CGT GGT CGC CAT Quencher-1 MUT Probe
67793 CCT GGA AGT TTA TTT ATT ACT AAC TCG Mutant Forward A
67794 AGA AGC CAT GTC CCC AGA Wild type Forward A
67795 TCT CAA ATG CAC GCC TTC Wild type Reverse A
67796 Fluorophore-2 ACA ACT GAG GCG TCT GAG TCC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
67619 0.40 uM
67793 0.40 uM
67794 0.40 uM
67795 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.