Stock Number: 002213 Strain Name: B6.129S4-Ngfrtm1Jae/J

Allele: Ngfrtm1Jae Protocol Name: Ngfrtm1Jae Method: Standard PCR Version: 3.1 Created: 15-September-2008 Updated: 29-October -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 = ~193 bp Heterozygote = ~193 bp and ~386 bp Wild type = ~386 bp

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

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR7617 - GCT CAG GAC TCG TGT TCT CC - Common oIMR7618 - CCA AAG AAG GAA TTG GTG GA - Wild type Reverse oIMR8162 - TGG ATG TGG AAT GTG TGC GAG - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.30 - 3.30 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 2.00 mM 0.96 20 uM oIMR7617 0.60 1.00 uM 0.60 20 uM oIMR7618 0.80 1.33 uM 0.80 20 uM oIMR8162 0.40 0.67 uM 0.40 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.12 0.01 U/ul 0.12 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 50 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 002124 C;129S-Ngfrtm1Jae/J 002213 B6.129S4-Ngfrtm1Jae/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.