Stock No: 002020
Protocol 21922: End Point Analysis Assay - Apc<Min>alternate1
Version 5.0

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 = A/A
Heterozygote = T/A
Wild type = T/T

amplicon = 85 bp


 

Sequence

GACATGATGATAGTAGGTCAGACAATTTCAATACTGGAAACATGACTGTTCTTTCACCATATTTAAATACTACGGTATTGCCCAGCTCTTCTTCCTCAAGGGGAAGTTTAGACAGTTCTCGTTCTGAGAAAGACAGAAGTT(t/a)GGAGAGAGAGCGAGGTATTGGCCTCAGTGCTTACCATCCAACAACAGAAAATGCAGGAACCTCATCAAAACGAGGTCTGCAGATCACTACCACTGCAGCCCAGATAGCCAAAGTTATGGAAGAAGTATCAGCCATTCATACCTCCCAGGACGACA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13779 GGG AAG TTT AGA CAG TTC TCG T Forward A
13780 TGT TGG ATG GTA AGC ACT GAG Reverse A
13781 Fluorophore-1 AGA CAG AAG TTA GGA GAG AGA GC Quencher-1 MUT Probe
13782 Fluorophore-2 AGA CAG AAG TTT GGA GAG AGA GC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
13779 0.40 uM
13780 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.