Stock Number: 006337 Strain Name: B6.Cg-Lgals1tm1Rob/J

Allele: Lgals1tm1Rob Protocol Name: Lgals1tm1Rob Method: Standard PCR Version: 1.1 Created: 25-June -2008 Updated: 24-September-2009

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). Expected Results: Mutant=~150 bp Heterozygote = ~150 bp and ~390 bp Wild type=~390 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 1.5% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR6918 - AAA CTT CAG CCG GGA GAA AGG - Common oIMR6919 - GAC CCC ATC CCT ACA CCC CAG - Wild type Forward oIMR8306 - CTA TCA GGA CAT AGC GTT GG - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.46 - 3.46 10 X PCR Buffer II (ABI) 0.96 0.80 X 0.96 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR6918 0.60 1.00 uM 0.60 20 uM oIMR6919 0.60 1.00 uM 0.60 20 uM oIMR8436 0.60 1.00 uM 0.60 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.20 0.02 U/ul 0.20 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 65 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 006337 B6.Cg-Lgals1tm1Rob/J 006354 B6.Cg-Lgals3tm1Poi Lgals1tm1Rob/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.