Protocol 31364: Probe Assay - Cdk17<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=  134 bp

Wild Type = 139 bp

>chr10:93216463+93216601 139bp GGATCTCAATGGAGGTAACCA GGCAAAGAAACAGCTCTTGTG

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36717 TGT CAG GTT GAC CCA AAA CA Mutant Forward A
36718 GGC AAA GAA ACA GCT CTT GTG Common A
36719 GGA TCT CAA TGG AGG TAA CCA Wild type Forward A
36720 Fluorophore-1 AGC ACA GCT GGC CTG AGA Quencher-1 MUT Probe
36721 Fluorophore-2 AGT GGA AAC GCA ACC ATT GT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36717 0.40 uM
36718 0.40 uM
36719 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.