Stock Number: 004189 Strain Name: B6.129S4-Prkcetm1Msg/J

Allele: Prkcetm1Msg Protocol Name: Prkcetm1Msg Method: Standard PCR Version: 1.1 Created: 20-August -2004 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 = 172 bp Heterozygote = 396 bp and 172 bp Wild type = 396 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 oIMR0158 - CTG AAT GAA CTG CAG GAC GA - Mutant oIMR0159 - ATA CTT TCT CGG CAG GAG CA - Mutant oIMR1577 - CCA CAA GGT GTA GCG AGT GA - Wild type oIMR1578 - CCG ATA GGA GCG TCT TGA AA - Wild type

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.22 - 3.22 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 oIMR1577 0.60 1.00 uM 0.60 20 uM oIMR1578 0.60 1.00 uM 0.60 20 uM oIMR0158 0.30 0.50 uM 0.30 20 uM oIMR0159 0.30 0.50 uM 0.30 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 1.5 min - 2 94 30 sec - 3 60 1 min - 4 72 45 sec 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 004189 B6.129S4-Prkcetm1Msg/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.