Protocol 30106: Probe Assay - Mylk4<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

>chr13:32816974-32817057 84bp TCCAGCCCCAGAAATATGAC CACAAACTCTTTGGGGTCAC

Mutant=  84 bp

Wild Type = 91 bp

Sequence

Wt Sequence: tccagccccagaaatatgactgccttGGaacttcagatttctcgctgttttgcattgtctaaatgtgaccccaaagagtttgtg

 

Mutant Sequence: tccagccccagaaatatgactgccttGGctttatttgccatgtcataaagtctgagagtaggaaaaacccagtgtgactcgccaaaaggtc

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
33917 TCC AGC CCC AGA AAT ATG AC Common A
33918 CAC AAA CTC TTT GGG GTC AC Wild type Reverse A
33919 GAC CTT TTG GCG AGT CAC AC Mutant Reverse A
33920 Fluorophore-1 CCT TGG AAC TTC AGA TTT CTC G Quencher-1 WT Probe
33922 Fluorophore-2 CCA TGT CAT AAA GTC TGA GAG TAG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
33917 0.40 uM
33918 0.40 uM
33919 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.