Protocol 42079: Probe Assay - Zfp622<em1(IMPC)J> Alternate 3
Version 4.0

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

Mut = 146 bp

Wt = 139 bp

Fam = Mut

Hex = Wt

>chr15:25991365+25991503 139bp CCTTTTAAGGGTTATTCGGTCA ACTAAACGGGAGACCTGCAT

Sequence

MUT Sequence (junction in uppercase):

ccttttaagggttattcggtcacatttgttggcagaaaaattctgacagtaatttttatcactcagttcttgtagtggGCaagactccatgacttaaaaaattaatattcctggtctagtaacttagtatgcatcatgaacctttccagaagata

This mutation is a 5037 bp deletion beginning at Chromosome 15 position 25,991,444 bp and ending after 25,996,480 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
56699 GGA AAG GTT CAT GAT GCA TAC TAA G Mutant Reverse A
59620 CCT TTT AAG GGT TAT TCG GTC A Common A
59621 ACT AAA CGG GAG ACC TGC AT Wild type Reverse A
59623 Fluorophore-1 ATT TGG TAG CTT GGC CAC ATT T Quencher-1 WT Probe
59814 Fluorophore-2 AGT GGG CAA GAC TCC ATG ACT TAA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
56699 0.40 uM
59620 0.40 uM
59621 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.