Stock Number: 003000 Strain Name: B6;129S-Apobtm2Sgy Ldlrtm1Her/J

Allele: Apobtm2Sgy Protocol Name: Apobtm2Sgy Method: Restriction Enzyme Digest Version: 1.1 Created: 02-May -2003 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 = 2500 bp and 300 bp Heterozygotes = 2800 bp and 2500 bp and 300 bp Wild type = 2800 bp (uncut)

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 oIMR1745 - TCC TGA GCT GCT GTA GTT TTT C - Common oIMR1776 - CCG CAT GAA GAC TGA CTC TG - Common

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 5.84 - 5.84 10 X PCR Buffer II (ABI) 1.20 1.00 X 1.20 25 mM MgCl2 1.20 2.50 mM 1.20 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR1776 0.30 0.50 uM 0.30 20 uM oIMR1745 0.30 0.50 uM 0.30 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 3 min - 2 94 30 sec - 3 65 1 min - 4 72 2 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 Hind III 0.80 - 0.80 This genotyping protocol is used for the following strains:   Stock Number Strain Name 002877 B6;129S7-Apobtm2Sgy/J 002879 B6;129-Apobtm2Sgy Apoetm1Unc/J 003000 B6;129S-Apobtm2Sgy Ldlrtm1Her/J 004192 STOCK Mttptm2Sgy Ldlrtm1Her Apobtm2Sgy Tg(Mx1-cre)1Cgn/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.