Stock No: 006013
Protocol 22064: End Point Analysis Assay - Tpo<tee>
Version 3.0

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

Sequence

GAGTTCTGTGGCTTGTCACGCCTGGAGACACCAGCTGAGCTGAACAAGGCCATTGCCAACAGAAGCATGGTCAACAAGATAATGGACTTAt/aACAAG
CATGCTGACAACATTGACGTCTGGTTGGGAGGCTTGGCTGAAAAGTTCTTGCCGGGGGCCCGCACTGGTCCTCTGTTTGCATGTATCATTGGGAAG
CAGATGAAGGCTCTG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
18550 GAG TTC TGT GGC TTG TCA CG Forward A
18551 CAG AGC CTT CAT CTG CTT CC Reverse A
18554 Fluorophore-1 ATA ATG GAC TTA TAC AAG CAT GCT G Quencher-1 WT Probe
18555 Fluorophore-2 ATA ATG GAC TTA AAC AAG CAT GCT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
18550 0.40 uM
18551 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.