Stock No: 016588
Protocol 21991: End Point Analysis Assay - Kit<W-v>Alternate1
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

122 bp

 

Sequence

GAGGCCCTAATGTCGGAACTGAAGGTCCTGAGCTACCTGGGCAATCACATGAATATTGTGAACCTGCTTGGCGCATGCAc/tGGTGGGAGgtgagtctcgagttgccatctctctggttatcaa

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
17087 TTG ATA ACC AGA GAG ATG GCA AC Reverse A
17089 Fluorophore-1 CGC ATG CAT GGT GGG AG Quencher-1 MUT Probe
23173 GAG GCC CTA ATG TCG GAA CT Forward A
23174 Fluorophore-2 CGC ATG CAC GGT GGG AG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
17087 0.40 uM
23173 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

Stock Number Strain Name
016588 A.B6-KitW-v/BeiMmjax
000049 C57BL/6J-KitW-v/J
100410 WBB6F1/J-KitW/KitW-v/J
3 strains use this protocol