Stock Number: 008469 Strain Name: STOCK Wnt9btm1.2Amc/J

Allele: Wnt9btm1.2Amc Protocol Name: Wnt9btm1.2Amc Method: Standard PCR Version: 1.1 Created: 09-February -2009 Updated: 25-November -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 = 300 bp Heterozygote = 219 bp and 300 bp Wild type = 219 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 oIMR9031 - GCA GAA TCT GGA GAA CTT GGC - Forward oIMR9032 - GTG AGA AGG AAG ATG GTG AGC - Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.90 - 3.90 10 X AB PCR BufferII 1.20 1.00 X 1.20 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR9031 0.60 1.00 uM 0.60 20 uM oIMR9032 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) 0.12 0.05 U/ul 0.12 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 66 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 008469 STOCK Wnt9btm1.2Amc/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.