Stock No: 028087
Protocol 22091: End Point Analysis Assay - Tuba4a<em#3(W407X)Lutzy>
Version 2.0

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 = A/A
Heterozygote = G/A
Wild type = G/G

Sequence

CAACACAACCGCCAttgctGAAGCCTGGGCCCGTCTGGACCACAAGTTTGACTTGATGTATGC
CAAGAGGGCTTTTGTACACTG(g/a)TATGTGGGTGAGGGAATGGAGGAGGGGGAGTTCTCTG
AGGCTCGAGAGGATATGGCTGCCCTGGAAAAAGATTATGA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
24785 ACA ACC GCC ATT GCT GAA G Forward A
24786 CCA TAT CCT CTC GAG CCT CA Reverse A
24787 Fluorophore-1 TTT TGT ACA CTG GTA TGT GGG TGA G Quencher-1 WT Probe
24788 Fluorophore-2 TTT TGT ACA CTG ATA TGT GGG TGA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
24785 0.40 uM
24786 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.