Protocol 31477: Probe Assay - Srsf9<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=  94 bp

Wild Type = 102 bp

>chr5:115330651+115330752 102bp TGTTTCAGGTATGTTCTCACCA CTCCTGAGAAGCCAGCATGT

Sequence

Wt Sequence:

 

Mutant Sequence:

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36964 TGT GTC TGG TGT CCA TCT CC Mutant Forward A
36965 CTC CTG AGA AGC CAG CAT GT Common A
36966 TGT TTC AGG TAT GTT CTC ACC A Wild type Forward A
36967 Fluorophore-1 CCC TCT GGC TGA CTT CCC Quencher-1 MUT Probe
36968 Fluorophore-2 ACG AGA GTC TAG CCT TCA AGG A Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36964 0.40 uM
36965 0.40 uM
36966 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.