Protocol 20029: End Point Analysis Assay - Frmd4b<tvrm222>-EP
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/C
Heterozygote = C/T
Wild type = T/T

Sequence

GGGAACTGGAGGACCCAGTTAACCATTGGGCTATCTGAATATGAAAATCCAGTGCATTCTCCCTACACCAGCTACTATGGCAG
CATCTACAATCCCTTG(t/c)CCTCTCCGAGCAGgtgagaaagtgttggccaggttctcggagcctggtcctctttgaaggacg
tgtggctcctacactgcagcctggtggtgaagaggcataaagtgggattt

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
29702 TCC AGT GCA TTC TCC CTA CA Forward A
29703 CTG GCC AAC ACT TTC TCA CC Reverse A
29704 Fluorophore-1 CAA TCC CTT GTC CTC TCC GA Quencher-1 WT Probe
29707 Fluorophore-2 ATC CCT TGC CCT CTC CG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
29702 0.40 uM
29703 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.