Protocol 21964: End Point Analysis Assay - Glra1<tm3.1Geh>
Version 3.0

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 = GCCGCC/GCCGCC
Heterozygote = AAGAAG/GCCGCC
Wild type = AAGAAG/AAGAAG

Sequence

CGCCTCCTGCGCCATCCAAGTCCCCGGAGGAGATGCGGAAACTCTTCAT

CCAGAGAGCC(AGGAGG/GGCGGC)ATCGACAAGAT(a/c)TCTCGCATCGG

TTTCCCCATGGCCTTCCTCATCTTCAACATGTTCTACTGGATCATCTA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
18234 GTG CCA ACA ACA ACA ACA CC Forward A
18235 ATC CTC TCT CCG GAC GAT CT Reverse A
18236 Fluorophore-1 AGA GAG CCA AGA AGA TCG ACA A Quencher-1 WT Probe
18237 Fluorophore-2 AGC CGC CGC CAT CG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
18234 0.40 uM
18235 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.