Stock No: 000632
Protocol 21994: End Point Analysis Assay - Lep<ob>
Version 5.0

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

Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13373 Fluorophore-1 GTG CTG CAG ATA GCC AAT GAC Quencher-1 Forward A
13374 Fluorophore-2 GAG AAG GCC AGC AGA TGG A Quencher-2 Reverse A
13375 Fluorophore-3 TGG AGA ATC TCC GAG ACC Quencher-3 WT Probe
13667 Fluorophore-4 CTG GAG AAT CTC TGA GAC C Quencher-4 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
13373 0.40 uM
13374 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
006906 B6.Cg-Lepob Ldlrtm1Her/J
000632 B6.Cg-Lepob/J
000696 BKS.Cg-Lepob/J
004824 BTBR.Cg-Lepob/WiscJ
4 strains use this protocol