Stock Number: 008695 Strain Name: FVB-Tg(tetO-MET)23Rwng/J

Allele: Tg(tetO-MET)23Rwng Protocol Name: Tg(tetO-MET) Method: Separated PCR Version: 1.1 Created: 13-February -2009 Updated: 24-September-2009

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Wild type = 750 bp Hemi = 200 bp and 550 bp Internal Control = 200 bp Transgene = 550 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 oIMR8744 - CAA ATG TTG CTT GTC TGG TG - Internal Positive Control Forward oIMR8745 - GTC AGT CGA GTG CAC AGT TT - Internal Positive Control Reverse oIMR9095 - GTC GAG TAG GCG TGT ACG - Forward oIMR9099 - GAA TGA CAT TCT GGA TGG GTG - Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O ###### - 478.00 10 X High Fidelity Buffer 1.20 1.00 X 1.20 50 mM MgSO4 0.96 4.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 uM oIMR9095 0.60 1.00 uM 0.60 20 uM oIMR9099 0.60 1.00 uM 0.60 DMSO 0.80 - 0.80 5 U/ul High Fidelity Platinum Taq 0.10 0.04 U/ul 0.10 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 56 45 sec - 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) ddH2O 4.78 - 4.78 10 X High Fidelity Buffer 1.20 1.00 X 1.20 50 mM MgSO4 0.96 4.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 20 oIMR8744 0.60 1.00 0.60 20 uM oIMR8745 0.60 1.00 uM 0.60 DMSO 0.80 - 0.80 5 U/ul High Fidelity Platinum Taq 0.10 0.04 U/ul 0.10 DNA 2.00 - 2.00 This genotyping protocol is used for the following strains:   Stock Number Strain Name 008695 FVB-Tg(tetO-MET)23Rwng/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 56 45 sec - 4 72 45 sec repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold