Protocol 21212: Probe Assay - Nipsnap2<em1J>
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

Mut= 125 bp

Wt= 130 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31966 GTA GAG GCA GTC GGC AGA AC Common A
31967 Fluorophore-1 ATG GCT CCC CTG TTT AAT CG Quencher-1 WT Probe
31968 TGG ATC TTT GGT AAC ACT TCT TGA C Wild type Reverse A
31969 ACA ACA TTG TAC TGA GCC TTG G Mutant Reverse A
31970 Fluorophore-2 TAC CAT GTT CAC GTG GTG GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
31966 0.40 uM
31968 0.40 uM
31969 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.