Stock Number: 006130 Strain Name: CBACa.Cg-ScribCrc/RachJ

Allele: ScribCrc Protocol Name: ScribCrc Method: Standard PCR Version: 1.1 Created: 09-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). Primers flank mutation (SNP). Genotype by sequencing PCR product (wild type allele has 7 C's, the mutant allele has 8 C's). Expected Results: Mutant = 183 bp Wild type = 183 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 oIMR7934 - CTG ACT GCT GCT TCC CCT AC - Forward oIMR7935 - TCC ACA GGG TAT GGT CCT TC - Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.62 - 3.62 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 oIMR7934 0.75 1.25 uM 0.75 20 uM oIMR7935 0.75 1.25 uM 0.75 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.10 0.01 U/ul 0.10 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 5 min - 2 94 30 sec - 3 58 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 006130 CBACa.Cg-ScribCrc/RachJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.