Protocol 30746: Probe Assay - Zfp706<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=  119 bp

Wild Type = 128 bp

>chr15:36933756-36933883 128bp CTGGCATTCCACTGTGTTATCT TCACAATGCATGCTCAACAG

Sequence

Wt Sequence:ctggcattccactgtgttatctgttTttaccaatgatatttgaactgtctggttttagaacaggttctgtaaacatccttttatacattgatgtacatacatttgatactgttgagcatgcattgtga

 

Mutant Sequence:ctggcattccactgtgttatctgttTGtaaaacagtatcctgccctttgtagaataaaggtgtccttgtggagtttataatcaattcactttggttgcattcttagtgtggctaaggct

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35349 CTG GCA TTC CAC TGT GTT ATC T Common A
35350 AGC CTT AGC CAC ACT AAG AAT G Mutant Reverse A
35351 TCA CAA TGC ATG CTC AAC AG Wild type Reverse A
35352 Fluorophore-1 AGA ATA AAG GTG TCC TTG TGG AGT T Quencher-1 MUT Probe
35353 Fluorophore-2 TGA ACT GTC TGG TTT TAG AAC AGG T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35349 0.40 uM
35350 0.40 uM
35351 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.