Stock Number: 006883 Strain Name: B6.Cg-Ldlrtm1Her Sod2tm1Leb/J

Allele: Sod2tm1Leb Protocol Name: Human HPRT Method: Standard PCR Version: 1.1 Created: 14-June -2001 Updated: 29-October -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). Use Perfect Match© (Stratagene) added before Taq to delete non-specific bands. Expected Results: Mutant (HPRT) = ~240 bp Heterozygote = ~123 bp and ~240 bp Wild type (Sod1) = ~123 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 oIMR0781 - TGT TCT CCT CTT CCT CAT CTC C - Mutant oIMR0782 - ACC CTT TCC AAA TCC TCA GC - Mutant oIMR0878 - TGA ACC AGT TGT GTT GTC AGG - Wild type oIMR0888 - TCC ATC ACT GGT CAC TAG CC - Wild type

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.50 - 3.50 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 oIMR0781 0.70 1.17 uM 0.70 20 uM oIMR0782 0.70 1.17 uM 0.70 20 uM oIMR0878 0.10 0.17 uM 0.10 20 uM oIMR0888 0.10 0.17 uM 0.10 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 1 U/ul Perfect Match (Stratagene) 0.06 0.01 U/ul 0.06 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.06 0.01 U/ul 0.06 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 20 sec - 3 61 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 001224 SOD1/EiJ 002968 B6;129S7-Mdm2tm1Bay/J 002972 B6;129S7-Sod1tm1Leb/J 002973 B6.129S7-Sod2tm1Leb/J 003277 B6;129S7-Acvr2atm1Zuk/J 003283 B6;129S7-Fshbtm1Zuk/J 003783 B6.129S7-Bmp7tm1Kry/J 003881 B6.129S7-Sod1tm1Leb/DnJ 006408 B6.Cg-Dab1scm-3J/J 006883 B6.Cg-Ldlrtm1Her Sod2tm1Leb/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.