Protocol 43367: Probe Assay - Msantd3<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

  
>chr4:48552312+48552416 105bp TCCTCTGTGACTTGTGACATTT GCATTTTCCACTCTATTTCTCC

Mutant=  80 bp

Wild Type = 105 bp

Sequence

Large deletion:  Mutant sequence with junction in uppercase

tcctctgtgacttgtgacattttgtagtattaGGgggttaggatttatacccactctcaccaacgtgtttggagatacgg

 

This mutation is a 9774 bp deletion beginning at Chromosome 4 position 48,552,345 bp and ending after 48,562,118 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
62922 TCC TCT GTG ACT TGT GAC ATT T Common A
62923 GCA TTT TCC ACT CTA TTT CTC C Wild type Reverse A
62924 CCG TAT CTC CAA ACA CGT TG Mutant Reverse A
62925 Fluorophore-1 TTT GCA GGC TAG CCA ACG A Quencher-1 WT Probe
62926 Fluorophore-2 AGT ATT AGG GGG TTA GGA TTT ATA CCC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
62922 0.40 uM
62923 0.40 uM
62924 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.