Protocol 36039: End Point Analysis Assay - Otc<em1Bshep>
Version 1.0

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 = C, A, A

Wild type = A, G, C

Sequence

 TCCTTTCTTACCACACAAGACATTCACTTGGGTGTGAATGAAAGTCTCAC(a/c)GACAC(c/a)GCTC(g/a)gtttgtaaaacttttcttccttccaaagtttatttcaaactc

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
18263 CAA GAC ATT CAC TTG GGT GTG Forward A
47661 TGA AAT AAA CTT TGG AAG GAA GA Reverse A
47662 Fluorophore-1 CAC AGA CAC CGC TCG GTT Quencher-1 WT Probe
47663 Fluorophore-2 CTC ACC GAC ACA GCT CAG TTT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
18263 0.40 uM
47661 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.