Stock Number: 004201 Strain Name: B6.Cg-Selplgtm1Fur/J

Allele: Selplgtm1Fur Protocol Name: Selplgtm1Fur Method: Standard PCR Version: 2.1 Created: 13-April -2009 Updated: 19-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 = ~250 bp Heterozygote = 140 bp and ~250 bp Wild type = 140 bp

Gel Image

Image Image file View Full Size

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

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1793 - AGC TTC CTT GTG CTG CTG AC - Wild type Forward oIMR1794 - TCA AAA TCG TCA TCC CCA AC - Common oIMR7412 - ATT AAG GGC CAG CTC ATT CC - Mutant

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.52 - 3.52 10 X PCR Buffer II (ABI) 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 oIMR1794 0.60 1.00 uM 0.60 20 uM oIMR1793 0.30 0.50 uM 0.30 20 uM oIMR7412 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 5 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 004201 B6.Cg-Selplgtm1Fur/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.