Protocol 43887: Probe Assay - Mt4<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

Mutant=  93 bp

Wild Type = 97 bp

  
>chr8:94136950+94137046 97bp ATGAGTGCCACGGGAACCT TGGTCGTTTAGGAGGCTTTC

Sequence

Large deletion:  Mutant sequence with junction in uppercase

atgagtgccacgggaacctcacctggcttccttaacACacgtggctgctcattgctgctcactttaaggcgctgcaactgtgcctggggcttt

 

This mutation is a 2150 bp deletion beginning at Chromosome 8 position 94,136,987 bp and ending after 94,139,136 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64240 ATG AGT GCC ACG GGA ACC T Common A
64241 TGG TCG TTT AGG AGG CTT TC Wild type Reverse A
64242 Fluorophore-1 CTA TGG CAG GGA AGC AGG T Quencher-1 WT Probe
64243 AAA GCC CCA GGC ACA GTT Mutant Reverse A
64244 Fluorophore-2 CTT CCT TAA CAC ACG TGG CTG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64240 0.40 uM
64241 0.40 uM
64243 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.