Stock Number: 004939 Strain Name: NOD/ShiLtJ-Leprdb-5J/LtJ

Allele: Leprdb-5J Protocol Name: Leprdb-5J Method: Restriction Enzyme Digest Version: 1.1 Created: 04-September-2008 Updated: 24-September-2009

Expected Results: Mutant = 250 bp Heterozygote = 100 bp, 190 bp and 250 bp Wild type = 100 bp and 190 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 oIMR6039 - ATA GAT GCG AGC ATC AGC AA - Common oIMR6040 - GGT AAT GAC AGG GCT ATG AAC A - Common

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.96 - 3.96 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 oIMR6039 0.60 1.00 uM 0.60 20 uM oIMR6040 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 0.06 0.03 U/ul 0.06 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 62 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold**

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) 10 X Buffer 1.20 - 1.20 ddH2O 6.40 - 6.40 Hae III 0.40 - 0.40 This genotyping protocol is used for the following strains:   Stock Number Strain Name 004939 NOD/ShiLtJ-Leprdb-5J/LtJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.