Protocol 33934: End Point Analysis Assay - Ighmbp2<nmd-2J>-EP-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 = G/G
Heterozygote = A/G
Wild type = A/A

Sequence

AGCTAAAGAGGTGCTGGGTGCTTAGGTGATGGTGTCCCTGAACTCGTGCCCCAAAGAGCTCCTGATTTTGGCTCTGGTCCTCACAGAGCACTGATGACACTGAAGAAGTACCACTCTGGGCCAGCATCCTCCCTCATAGACATCCTGTTGGGCAGCTCCACCCCAAGTCCTGCCATGGAAATACGTGAGGATTTTTAATTTTCTCA(a/g)GTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGGGAGAGAGAGAGAGAGATGGAGGGGGAGGGAGGGAGAGAGAGAGATGGAGGGGGAGGGAGGGAGAGAGAGAGAGAGAATGAATATGTATCCACATGAGT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41462 GCC ATG GAA ATA CGT GAG GA Forward A
41464 Fluorophore-1 TTT TAA TTT TCT CAA GTA TGT GTG TGT GT Quencher-1 WT Probe
41465 Fluorophore-2 TTT AAT TTT CTC AGG TAT GTG TGT GTG Quencher-2 MUT Probe
42509 CCT CCC TCC CCC TCC ATC T Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41462 0.40 uM
42509 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.