Protocol 30718: Probe Assay - Scg3<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

>chr9:75524241-75524345 105bp AGCACACACACCCATCACAC TCCCTCTGAAGTCTTTCCTGA

 

Mutant=  136 bp

Wild Type = 105 bp

Sequence

Wt Sequence: agcacacacacccatcacacaagctgcttgagagtcagaggagctgcgcagcttgccagggaatgtctaGTgcccagctggaagtcaggaaagacttcagaggga

 

Mutant Sequence: gccaggctgacatagtaggatgctatctctaaatagatggatgataaattaattaattaaatgcaatgaaaaagttaagtatttcaaaaagggataggttATgcccagctggaagtcaggaaagacttcagaggga

406 bp deletion beginning at Chromosome 9 negative strand position 75,676,874 bp, TGAAATGATGCGTGGACTAG, and ending after GCTTGCCAGGGAATGTCTAG at 75,676,469 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35270 Fluorophore-1 AGG GAT AGG TTA TGC CCA GC Quencher-1 MUT Probe
35271 AGC ACA CAC ACC CAT CAC AC Wild type Forward A
35272 TCC CTC TGA AGT CTT TCC TGA Common A
35273 GCC AGG CTG ACA TAG TAG GA Mutant Forward A
35274 Fluorophore-2 AGG AGC TGC GCA GCT T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35271 0.40 uM
35272 0.40 uM
35273 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.