Protocol 39788: End Point Analysis Assay - Scimp-rs61481506-EP-alt1
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 = G/G
Heterozygote = A/G
Wild type = A/A

>chr11:70822500-70822604 105bp CCCCTCCAGTCAGTGAGTATCT TAGCTCTGGCTGGCTGACTG

The Rev primer (primer 54332) anneals over the nucleotide sequence containing mouse genomic variation rs225759573 and the WT and Mut probes (primer 54333 and 54334) anneal over the nucleotide sequence containing mouse genomic variation rs259100961.

Sequence

GAACTTGTACTCTGAGGGCCCCTCCAGTCAGTGAGTATCTGGATTTGCAAGACAGGCAAGAGGACA(a/g)GTCCTGGTCTCTTGCCCTAACACACAGGCAATCAGCCAGTCAGCCAGCCAGAGCTATATTTAGATTTCTGTCAT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
54331 CCC CTC CAG TCA GTG AGT ATC T Forward A
54332 TAG CTC TGG CTG GCT GAC TG Reverse A
54334 Fluorophore-1 CAA GAG GAC AGG TCC TGG TCT Quencher-1 MUT Probe
54624 Fluorophore-2 AAG AGG ACA AGT CCT GGT CT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
54331 0.40 uM
54332 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.