Stock Number: 004155 Strain Name: B6.129S2-Alox5tm1Fun/J

Allele: Alox5tm1Fun Protocol Name: Alox5tm1Fun Method: Standard PCR Version: 1.1 Created: 23-January -2001 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 = 600 bp Heterozygote = 164 bp and 600 bp Wild type = 164 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 oIMR0510 - ATC GCC TTC TTG ACG AGT TC - Mutant oIMR0511 - GCA GGA AGT GGC TAC TGT GGA - Common oIMR0512 - TGC AAC CCA GTA CTC ATC AAG - Wild type

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.98 - 2.98 10 X PCR Buffer II (ABI) 1.20 1.00 X 1.20 25 mM MgCl2 1.20 2.50 mM 1.20 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR0510 0.60 1.00 uM 0.60 20 uM oIMR0511 0.60 1.00 uM 0.60 20 uM oIMR0512 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 68 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 002263 B6;129S2-Alox5tm1Fun/J 002485 129-Alox5tm1Fun/J 004155 B6.129S2-Alox5tm1Fun/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.