Genotyping Protocols Database
Stock No: 006337
Protocol 26048: Standard PCR Assay - Lgals1<tm1Rob>
Version 3.2

Notes

This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase).
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=~250 bp
Heterozygote = ~250 bp and ~390 bp
Wild type=~390 bp

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
oIMR6918 AAA CTT CAG CCG GGA GAA AGG Common A
oIMR6919 GAC CCC ATC CCT ACA CCC CAG Wild type Forward A
oIMR8306 CTA TCA GGA CAT AGC GTT GG Mutant Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR6918 0.50 uM
oIMR6919 0.50 uM
oIMR8306 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
006337 B6.Cg-Lgals1tm1Rob/J
006354 B6.Cg-Lgals3tm1Poi Lgals1tm1Rob/J
2 strains use this protocol