Stock Number: 002020 Strain Name: C57BL/6J-ApcMin/J

Allele: ApcMin Protocol Name: ApcMin Method: Standard PCR Version: 3.1 Created: 18-July -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 = ~340 bp Heterozygote = ~340 bp and ~600 bp Wild type = ~600 bp

Gel Image

Image Image file View Full Size

Gel Information: Separated by gel electrophoresis on a 1.0% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR0033 - GCC ATC CCT TCA CGT TAG - Wild type oIMR0034 - TTC CAC TTT GGC ATA AGG C - Common oIMR0758 - TTC TGA GAA AGA CAG AAG TTA - Mutant

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 1.78 - 1.78 10 X PCR Buffer II (ABI) 1.55 1.29 X 1.55 25 mM MgCl2 1.15 2.40 mM 1.15 2.5 mM dNTP 1.15 0.24 mM 1.15 20 uM oIMR0033 0.89 1.48 uM 0.89 20 uM oIMR0034 0.90 1.50 uM 0.90 20 uM oIMR0758 0.72 1.20 uM 0.72 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 55 30 sec - 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 002020 C57BL/6J-ApcMin/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.