Stock No: 021999
Protocol 15710: End Point Analysis Assay - Tyr<em3J>
Version 2.0

Notes

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 = ------------------------
Heterozygote = agggaccactattacgtaatcctg/------------------------
Wild type = agggaccactattacgtaatcctg

 

 

Sequence

TCAGAAGAGTATAATAGCCATCAGGTTTTATGCGATGGAACACCTGagggaccactattacgtaatcctg/------------------------GAAACCATGACAAAGCCAAAACCCCCAGGCTCCCATCTTCAGCAGATGTGGAATTTTGTCTGAGTTTGACCCAGTATGAATCTGGATCAATGGATAGAACTGCCAATTTCAGC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
18428 TCA GAA GAG TAT AAT AGC CAT CAG G Forward
18429 GCT GAA ATT GGC AGT TCT ATC C Reverse
18430 Fluorophore-1 AGG GAC CAC TAT TAC GTA ATC CTG Quencher-1 WT Probe
18431 Fluorophore-2 GGA ACA CCT GGA AAC CAT GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 0.42
ddH2O
18428 0.17
18429 0.17
18430 0.08
18431 0.08
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 92.0 --
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.