Protocol 30731: Probe Assay - Tssk4<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=  95 bp

Wild Type = 102 bp

>chr14:56270095+56270196 102bp TGTATCCACTGTGTGAGAGCAC AAAGCCATGGAGTTGTGAGC

Sequence

Wt Sequence:tgtatccactgtgtgagagcactttatacacaatggtgaactagtggtcctcaggtaccatcctctctcccttttgggCtctgctcacaactccatggcttt

 

Mutant Sequence:CCACGTGAGATACAGgtcagaaagggagctgaaagaaggaacttaggcttattaataagctacttgaggtACtctgctcacaactccatggcttt

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35305 CCA CGT GAG ATA CAG GTC AGA Mutant Forward A
35306 AAA GCC ATG GAG TTG TGA GC Common A
35307 TGT ATC CAC TGT GTG AGA GCA C Wild type Forward A
35308 Fluorophore-1 AGC TGA AAG AAG GAA CTT AGG CTT A Quencher-1 MUT Probe
35309 Fluorophore-2 CAG GTA CCA TCC TCT CTC CCT T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35305 0.40 uM
35306 0.40 uM
35307 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.