Stock No: 009089
Protocol 21797: Probe Assay - Ndn<tm2Stw/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=  106 bp

Wild Type = 110 bp

Sequence

Wt Sequence: ACTGTGAGATGCAGGACAGCGATGCCGTTCCGGTGGGGATCCCtCCTCCCGCTTCTCTGGCCGCTAACCTCGCAGGGCCACCGTGCGCTCCCGAAGGCCCTATGGCAGCC

 

Mutant Sequence: ACTGTGAGATGCAGGACAGCGATGCCGTTCCGGTGGGGGATCCCgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagc

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
14989 GCT GCA AGG CGA TTA AGT TG Mutant Reverse A
33270 ACT GTG AGA TGC AGG ACA GC Common A
33271 GGC TGC CAT AGG GCC TTC Wild type Reverse A
33272 Fluorophore-1 TCG TGA CTG GGA AAA CCC T Quencher-1 MUT Probe
33273 Fluorophore-2 AAC CTC GCA GGG CCA C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
14989 0.40 uM
33270 0.40 uM
33271 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.