Protocol 30720: Probe Assay - Stk35<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

>chr2:129626907+129627017 111bp ACGGATAAGTCGGCGAGAG GAATTGAACGGGGGAAGG

Mutant=  116 bp

Wild Type = 111 bp

Sequence

Wt Sequence: acggataagtcggcgagaggggacggtttgcgcttcggtttccggtcctccctcgGTacttcccggtggttccgtctttcccgctttcctttcccttcccccgttcaattc

 

Mutant Sequence: acggataagtcggcgagaggggacggtttgcgcttcggtttccggtcctccctcgGTggtgggattcaaagtagttggtagaactgcagaagccccctcatgcctctttctcatca

1047 bp deletion beginning at Chromosome 2 positive strand position 129,801,227 bp TACTTCCCGGTGGTTCCGTC, and ending after AGATGGATGAAGAGTAGTCC at 129,802,273 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35278 ACG GAT AAG TCG GCG AGA G Common A
35279 GAA TTG AAC GGG GGA AGG Wild type Reverse A
35280 TGA TGA GAA AGA GGC ATG AGG Mutant Reverse A
35281 Fluorophore-1 CCG GTG GTT CCG TCT TT Quencher-1 WT Probe
35282 Fluorophore-2 CGG TGG TGG GAT TCA AAG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35278 0.40 uM
35279 0.40 uM
35280 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.