Stock No: 030811
Protocol 21671: End Point Analysis Assay - Fig4<em2(I41T)Lutzy>
Version 1.0

Notes

caaaatgcctttaaaatgacattctctttctttatagAGATACTTTCTAGTTGGGAGCAATCATGCAGAAACGAAGTACCGTGT(c/t)CTG
AAAA(t/c)TGATAGAACAGAACCGAAGGACTTGGTGGTAATCGACGACAGGgtgagtatcgttaggctccctgcccggccctcatgca
gggc
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, C
Heterozygote = C/T and T/C
Wild type = C, T

Sequence

caaaatgcctttaaaatgacattctctttctttatagAGATACTTTCTAGTTGGGAGCAATCATGCAGAAACGAAGTACCGT
GT(c/t)CTGAAAA(t/c)TGATAGAACAGAACCGAAGGACTTGGTGGTAATCGACGACAGGgtgagtatcgttaggctccctg cccggccctcatgcagggc
 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32990 GCA ATC ATG CAG AAA CGA AG Forward A
32991 TGT CGT CGA TTA CCA CCA AG Reverse A
32992 Fluorophore-1 CGT GTC CTG AAA ATT GAT AGA ACA G Quencher-1 WT Probe
32993 Fluorophore-2 CGT GTT CTG AAA ACT GAT AGA ACA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32990 0.40 uM
32991 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.