Protocol 45303: Probe Assay - Tti1<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

>chr2:158000978-158001057 80bp CTGGACATTCGAGAGCGATG TGCTGTTTTTCAGTGGCTTT

Mutant=  110 bp

Wild Type = 80 bp

The common forward primer (primer 65238) anneals over the nucleotide sequence containing mouse genomic variations rs27309128 and rs218935467.

Sequence

Wt Sequence: ctggacattcgagagcgatgtataagagttgtcctGaggcgggaaagtgcctcacctgctaaagccactgaaaaacagca

Mut Sequence:  ctggacattcgagagcgatgtataagagttgtcctGCaagagggagctgagtcccctaccttgaaaactgtgaagcctagtgggttcactgagtccccaaggcaaagagt

A 4131 bp deletion beginning at Chromosome 2 position 157,996,891 bp and ending after 158,001,021 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65238 CTG GAC ATT CGA GAG CGA TG Common A
65239 TGC TGT TTT TCA GTG GCT TT Wild type Reverse A
65241 ACT CTT TGC CTT GGG GAC TC Mutant Reverse A
65243 Fluorophore-1 TCC TGC AAG AGG GAG CTG A Quencher-1 MUT Probe
65244 Fluorophore-2 CGG GAA AGT GCC TCA CCT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65238 0.40 uM
65239 0.40 uM
65241 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.