Protocol 30744: Probe Assay - Zfp407<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=  113 bp

Wild Type = 103 bp

>chr18:84722065-84722167 103bp GAAAATTATGCGTGCGTATCC TGCTCATCTTCAGCACAGGA

Sequence

Wt Sequence:gaaaattatgcgtgcgtatcctccttccctttGttatggtaaattccttaaatgcagttatgacattcttctcccacttctaatcctgtgctgaagatgagca

 

Mutant Sequence:ttgagaattgggaagactcactaaaacattaaaactgtatgGGttatggtaaattccttaaatgcagttatgacattcttctcccacttctaatcctgtgctgaagatgagca

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35337 TTG AGA ATT GGG AAG ACT CAC T Mutant Forward A
35338 TGC TCA TCT TCA GCA CAG GA Common A
35339 GAA AAT TAT GCG TGC GTA TCC Wild type Forward A
35340 Fluorophore-1 TGG GTT ATG GTA AAT TCC TTA AAT G Quencher-1 MUT Probe
35341 Fluorophore-2 CTT CCC TTT GTT ATG GTA AAT TCC T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35337 0.40 uM
35338 0.40 uM
35339 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.