Stock Number: 008250 Strain Name: STOCK Tg(Ins2-rtTA)2Efr/J

Allele: Tg(Ins2-rtTA)2Efr Protocol Name: Tg(Ins2-rtTA)2Efr Method: Standard PCR Version: 1.1 Created: 11-July -2008 Updated: 24-September-2009

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene = ~430 bp Internal positive control = 200 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 oIMR8107 - TAG ATG TGC TTT ACT AAG TCA TCG CG - Transgene Forward oIMR8108 - GAG ATC GAG CGG GCC CTC GAT GGT AG - Transgene Reverse oIMR8744 - CAA ATG TTG CTT GTC TGG TG - Internal Positive Control Forward oIMR8745 - GTC AGT CGA GTG CAC AGT TT - Internal Positive Control Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.36 - 3.36 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 oIMR8107 0.60 1.00 uM 0.60 20 uM oIMR8108 0.60 1.00 uM 0.60 20 oIMR8744 0.30 0.50 0.30 20 uM oIMR8745 0.30 0.50 uM 0.30 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase 0.06 0.03 U/ul 0.06 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 50 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 008250 STOCK Tg(Ins2-rtTA)2Efr/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.