Stock No: 028092
Protocol 22067: End Point Analysis Assay - Tuba4a<em#1(R320C)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 = T/T
Heterozygote = C/T
Wild type = C/C

Sequence

GAGCAGCTGTCAGTGGCAGAGATCACCAATGCCTGTTTTGAGCCTGCCAACCAGATGGTGAAA
TGTGACCCTCGGCACGGCAAGTACATGGCCTGCTGCCTGCTGTAC(c/t)GTGGAGATGTGGTGC
CCAAAGATGTGAATGCTGCCATAGCTGCCATCAAGACGAAGCGCAGCATCCAGTTTGTGGACT
GGTGCCCCACAGG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
24794 TCA CCA ATG CCT GTT TTG AG Forward A
24795 TCG TCT TGA TGG CAG CTA TG Reverse A
24796 Fluorophore-1 CCT GCT GTA CCG TGG AGA TGT Quencher-1 WT Probe
24800 Fluorophore-2 GCC TGC TGT ACT GTG GAG ATG TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
24794 0.40 uM
24795 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.