Protocol 30885: Probe Assay - Cdh7<em1(IMPC)J> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

>chr1:110048639+110048829 191bp TTTCTCAGAGCTGGATTTCACA CATCAGAGTGGAGCTGGTGA

Mutant=  177 bp

Wild Type = 191 bp

Sequence

Wt Sequence: tttctcagagctggatttcacatTGtatattctggcttcttagcacaatcagcacttagctcatctttccaaaaacaatgaaatttcttacagctgtgacatgaaagtgatttttacataactttatattgctgactctcttgtaagttaatctcatctcacatgaatttttcaccagCTCCACTCTGATG

 

Mutant Sequence: tttctcagagctggatttcacatTgtgcaggcgtggtcagaaaggTtttccttttaacaatactcacttctttaaaattatgattacaataatctctaacaagttactattttgtttttaaaaacgtacttccctgtaaattcttccagctgtccctccatcccctctctcccta

355 bp of flanking intronic sequence including the splice acceptor and donor. Additionally, 55 bp after the end of this exon there is 21 bp of retained endogenous sequence (GTGCAGGCGTGGTCAGAAAGG) followed by an additional 144 bp deletion

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
33475 CAT CAG AGT GGA GCT GGT GA Wild type Reverse A
35748 TTT CTC AGA GCT GGA TTT CAC A Common A
35749 ACT AGG GAG AGA GGG GAT GG Mutant Reverse A
35750 Fluorophore-1 AGC ACA ATC AGC ACT TAG CTC AT Quencher-1 WT Probe
35752 Fluorophore-2 ACG TAC TTC CCT GTA AAT TCT TCC A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
33475 0.40 uM
35748 0.40 uM
35749 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.