Stock No: 002854
Protocol 22010: End Point Analysis Assay - Nek1<kat-2J> Endpoint
Version 3.0

Notes

Mutant = X bp
Heterozygote = X bp and Y bp
Wild type = Y bp

Converting to 12ul, email genotyping@jax.org with jpeg

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 = X bp
Heterozygote = X bp and Y bp
Wild type = Y bp

Sequence

gttgtctaatagaggtaactggactgaaacaatatttctgtttgtttattctttctttttcttatagAACATATTTCTAACCAAAGATGGGgACAGTGCAGCTTGGAGATTTTGGAATTGCTCG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
16868 CGA GCA ATT CCA AAA TCT CC Reverse A
16869 TTG TCT AAT AGA GGT AAC TGG ACT GA Forward A
16870 Fluorophore-1 AAA GAT GGG GAC AGT GCA G Quencher-1 MUT Probe
16871 Fluorophore-2 AAA GAT GGG ACA GTG CAG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
16868 0.40 uM
16869 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 4.0 -- 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.