Stock No: 034972
Protocol 39533: End Point Analysis Assay - Kcnq2<em6Lutzy>-alternate1
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 = GTTC

Wild type = ACAG

>chr2:181113013-181113102 90bp CTGGAAGCTCTTGGGATCG CTTGAAGTCATGGTAAACAGCAG

The For primer (primer 54043) anneals over the nucleotide sequence containing mouse genomic variations rs27642401 and the Rev primer (primer 54044) anneals over the nucleotide sequence containing mouse genomic variation rs227134979.

Sequence

ATCTTGCGGATGATCCGTATGGACCGGAGGGGTGGCACCTGGAAGCTCTTGGGATCGGTAGTCTACGCTC(a/g)(c/t)(a/t)(g/c)CAAGGTGAGCAGCACTGACCCAGCCTGGAGCCTGCTGTTTACCATGACTTCAAGGCTCCAGGCTGCTCTA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53556 Fluorophore-1 CTC ACA GCA AGG TGA GCA GC Quencher-1 WT Probe
53557 Fluorophore-2 CTC GTT CCA AGG TGA GCA GC Quencher-2 MUT Probe
54043 CTG GAA GCT CTT GGG ATC G Forward A
54044 CTT GAA GTC ATG GTA AAC AGC AG Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
54043 0.40 uM
54044 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.