Stock Number: 002465 Strain Name: B6;129S-Ldlrtm1Her Lrpap1tm1Her/J

Allele: Lrpap1tm1Her Protocol Name: Lrpaptm1Her Method: Standard PCR Version: 1.1 Created: 15-January -2001 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 = 280 bp Heterozygote = 78 bp and 280 bp Wild type = 78 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 oIMR0690 - GCT AGT GCT GTT GTT GCT GC - Wild type oIMR0691 - TTC TCT CGC GAG TAC TTG CC - 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 2.62 - 2.62 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 oIMR6916 0.60 1.00 uM 0.60 20 uM oIMR6917 0.60 1.00 uM 0.60 20 uM oIMR0690 0.60 1.00 uM 0.60 20 uM oIMR0691 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 + 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 20 sec - 3 64 30 sec* *-0.5 C per cycle 4 72 35 sec repeat steps 2-4 for 12 cycles 5 94 20 sec - 6 58 30 sec - 7 72 35 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 002464 B6;129S7-Lrpap1tm1Her/J 002465 B6;129S-Ldlrtm1Her Lrpap1tm1Her/J 002987 B6.129S7-Lrpap1tm1Her/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.