Protocol 31368: Probe Assay - Adgrf3<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

Mutant=  118 bp

Wild Type = 127 bp

>chr5:30199307-30199433 127bp CTCTCGTGGCTCCTGTCTCT GGCTCAAGGTACAGGGATAACA

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36727 CTC TCG TGG CTC CTG TCT CT Common A
36728 CAC CAC CAC CAC CAC ATA GA Mutant Reverse A
36729 GGC TCA AGG TAC AGG GAT AAC A Wild type Reverse A
36730 Fluorophore-1 AGT GGA TCC TCC AGG GAC A Quencher-1 MUT Probe
36733 Fluorophore-2 CTG ACC TCT GAG TGG CTT GTC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36727 0.40 uM
36728 0.40 uM
36729 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.