Protocol 30558: Probe Assay - Tpk1<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

>chr6:43510188-43510279 92bp GGGAAAAAGGTAGAATGTAGAGCA ACCACTCACAGACCCTTTGC

Mutant=  127 bp

Wild Type = 92 bp

Sequence

Wt Sequence: gggaaaaaggtagaatgtagagcaggtagctccatatcttctccaccTCtgttcagatggactggagaagatgcaaagggtctgtgagtggt

 

Mutant Sequence: gggaaaaaggtagaatgtagagcaggtagctccatatcttctccaccTGggctgtataaccatgatgcattacttctttctatgaatgagatctcttagaattaggatcatgttttgtcttgttttt

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34887 Fluorophore-1 TGG GCT GTA TAA CCA TGA TGC Quencher-1
34888 GGG AAA AAG GTA GAA TGT AGA GCA Common A
34889 ACC ACT CAC AGA CCC TTT GC Wild type Reverse A
34890 AAA AAC AAG ACA AAA CAT GAT CC Mutant Reverse A
34891 Fluorophore-2 CTG TTC AGA TGG ACT GGA GAA GA Quencher-2

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34888 0.40 uM
34889 0.40 uM
34890 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.