Stock Number: 003692 Strain Name: B6;129X1-Sncatm1Rosl/J

Allele: Sncatm1Rosl Protocol Name: Sncatm1Rosl Method: Standard PCR Version: 2.1 Created: 29-July -2008 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). Use Perfect Match© (Stratagene) added before Taq to delete non-specific bands. Expected Results: Mutant = 172 bp Heterozygote = 172 bp and 320 bp Wild type = 320 bp

Gel Image

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Gel Information: Separated by gel electrophoresis on a 3.0% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR0158 - CTG AAT GAA CTG CAG GAC GA - Mutant oIMR0159 - ATA CTT TCT CGG CAG GAG CA - Mutant oIMR1286 - GGC GAC GTG AAG GAG CCA GGG A - Wild type oIMR1287 - CAG CGA AAG GAA AGC CGA GTG ATG TAC T - Wild type

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 oIMR0158 0.60 1.00 uM 0.60 20 uM oIMR0159 0.60 1.00 uM 0.60 20 uM oIMR1286 0.60 1.00 uM 0.60 20 uM oIMR1287 0.60 1.00 uM 0.60 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 1 U/ul Perfect Match (Stratagene) 0.10 0.01 U/ul 0.10 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.10 0.01 U/ul 0.10 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 58 30 sec - 4 72 30 sec 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 003692 B6;129X1-Sncatm1Rosl/J 905068 B6;129X1-Sncatm1Rosl Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.