Protocol 31370: Probe Assay - Shtn1<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=  110 bp

Wild Type = 144 bp

  

>chr19:59042027-59042170 144bp GGTTTGTAGTTTGCATGTGTGC CCCCATGACCAAGAAGAATGT

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36741 GGT TTG TAG TTT GCA TGT GTG C Common A
36742 TGC TTA CAG AAT CAT ATT CCA GGA C Mutant Reverse A
36743 CCC CAT GAC CAA GAA GAA TGT Wild type Reverse A
36744 Fluorophore-1 ATG GGG AGG TGT AAG GTT CC Quencher-1 MUT Probe
36745 Fluorophore-2 AGC CAA AGG TCT AGC TGT AGG A Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36741 0.40 uM
36742 0.40 uM
36743 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.