Genotyping Protocols Database
Protocol 34825: End Point Analysis Assay - Plcg2<em3Adiuj>
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 = T, T

Wild type = A, C

Sequence

 

ACAATGACCACCATGGTCAACGTGGACACCCTTCCAGAATATGAGAAGAGTCAGATCAAGAGAGCGCTGGAGTTAGGGACGGTG(a/t)TGAC(c/t)GTGTTCAACGCCCGCAAATCCACCCCAGAGCGGAGGACAGTACAGATGATCATGGAGACTCGGCAGGT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44584 GAT CAA GAG AGC GCT GGA GT Forward A
44586 TGT ACT GTC CTC CGC TCT GG Reverse A
44588 Fluorophore-1 ACG GTG ATG ACC GTG TTC A Quencher-1 WT Probe
44589 Fluorophore-2 ACG GTG TTG ACT GTG TTC AAC G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44584 0.40 uM
44586 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 4.0 -- 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.