Protocol 35877: Probe Assay - Zfp423<em1(IMPC)J>Alternate1
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

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

>chr8:87780057-87780161 105bp CTTGGGGACCAGTGGGTATC CCTAACTCACACGGGCAGT

Mut= 120 bp

Wt= 105 bp

Fam=Mut

Hex=Wt

Sequence

Mut Sequence:

cctgggaattgcctctaatctctcctctgccttcccctgcagATGGTGATGACGACCCACAGCTCTCCTGGgtaggtcttatcgcaacccagagaggcagcactgcccgtgtgagttagg

This mutation is a 3279 bp deletion beginning at Chromosome 8 position 87,780,106 bp and ending after 87,783,384 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45658 CTT GGG GAC CAG TGG GTA TC Wild type Forward A
45659 CCT AAC TCA CAC GGG CAG T Common A
45661 Fluorophore-1 AAC TCT TGC ATG TGT GCT TCC Quencher-1 WT Probe
47218 CCT GGG AAT TGC CTC TAA TCT Mutant Forward A
47219 Fluorophore-2 CAC AGC TCT CCT GGG TAG GTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45658 0.40 uM
45659 0.40 uM
47218 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.