Stock Number: 006906 Strain Name: B6.Cg-Lepob Ldlrtm1Her/J

Allele: Lepob Protocol Name: Lepob Method: Restriction Enzyme Digest Version: 1.1 Created: 02-May -2003 Updated: 24-September-2009

Expected Results: Mutant = 55 bp and 100 bp Heterozygote = 55 bp, 100 bp and 155 bp Wild type = 155 bp

Gel Image

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Gel Information: Separate digested products on a 3.0% agarose gel.**

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1151 - TGT CCA AGA TGG ACC AGA CTC - Common oIMR1152 - ACT GGT CTG AGG CAG GGA GCA - 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 0.89 X 1.20 25 mM MgCl2 0.96 1.78 mM 0.96 2.5 mM dNTP 0.96 0.18 mM 0.96 20 uM oIMR1151 0.60 0.89 uM 0.60 20 uM oIMR1152 0.60 0.89 uM 0.60 5 mM DNA Loading Dye 1.66 0.62 mM 1.66 5 U/ul Taq DNA Polymerase 0.06 0.02 U/ul 0.06 DNA 3.50 - 3.50

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 62 1 min - 4 72 45 sec 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 6.18 - 6.18 10 mg/ml BSA 0.12 - 0.12 Dde I 0.50 - 0.50 This genotyping protocol is used for the following strains:   Stock Number Strain Name 000632 B6.V-Lepob/J 000696 BKS.V-Lepob/J 004824 BTBR.V(B6)-Lepob/WiscJ 006906 B6.Cg-Lepob Ldlrtm1Her/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.