Protocol 38113: Probe Assay - Cmtm8<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

>chr9:114789286-114789396 111bp CCTTCTAGCCTTTATTTTTCTTACAC CCACACACACGAAACAAATG

Mut = 82 bp

Wt = 111 bp

Fam = Mut

Hex = Wt

Common primer anneals over the nucleotide sequence containing mouse genomic variation rs248217201.

 

Sequence

MUT Sequence (junction in uppercase):

gtttttattatgtatattggggatatgtctacatgggtgggcatgtgtgtataatatgtacaggtTGagattacatttgtttcgtgtgtgtggatgtgtgtttgtgcatgggtgcgggggcgccaatgtgcc

This mutation is a 6990 bp deletion beginning at Chromosome 9 position 114,789,313 bp and ending after 114,796,302 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53026 CCA CAC ACA CGA AAC AAA TG Common A
53027 CCT TCT AGC CTT TAT TTT TCT TAC AC Wild type Forward A
53028 GTA TAT TGG GGA TAT GTC TAC ATG G Mutant Forward A
53029 Fluorophore-1 CGT GTG GAG TTT GTA AAG CCA Quencher-1 WT Probe
53030 Fluorophore-2 TGG GCA TGT GTG TAT AAT ATG TAC AG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
53026 0.40 uM
53027 0.40 uM
53028 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.