Stock Number: 002466 Strain Name: B6;129P2-Prkcctm1Stl/J

Allele: Prkcctm1Stl Protocol Name: Pkcctm1Stl Method: Standard PCR Version: 1.1 Created: 13-March -2001 Updated: 24-September-2009

Expected Results: Mutant = 280 bp Heterozygote = 154 bp and 280 bp Wild type = 154 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 oIMR0496 - GCT CCG ACG AAC TCT ATG CCA - Wild type oIMR0497 - GTG GAG TGA AGC TGC GTG AGA - Wild type oIMR6916 - CTT GGG TGG AGA GGC TAT TC - Mutant Forward oIMR6917 - AGG TGA GAT GAC AGG AGA TC - Mutant Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.05 - 3.05 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 oIMR6916 0.45 0.75 uM 0.45 20 uM oIMR6917 0.45 0.75 uM 0.45 20 uM oIMR0496 0.60 1.00 uM 0.60 20 uM oIMR0497 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 0.07 0.03 U/ul 0.07 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 1.5 min - 2 94 35 sec - 3 64 45 sec* *-0.5 C per cycle 4 72 45 sec repeat steps 2-4 for 12 cycles 5 94 35 sec - 6 58 30 sec - 7 72 45 sec repeat steps 5-7 for 25 cycles 8 72 2 min - 9 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 002466 B6;129P2-Prkcctm1Stl/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.