Stock Number: 007176 Strain Name: B6.Cg-Tg(Pax8-rtTA2S*M2)1Koes/J

Allele: Tg(Pax8-rtTA2S*M2)1Koes Protocol Name: Tg(Pax8-rtTA2S*M2)1Koes Method: Melt Curve Analysis Version: 3.1 Created: 30-April -2009 Updated: 24-September-2009

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene Tm = 87°C +/- 1.0°C Internal positive control Tm = 80°C+/- 1.0°C

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR7338 - CTA GGC CAC AGA ATT GAA AGA TCT - Internal Positive Control Forward oIMR7339 - GTA GGT GGA AAT TCT AGC ATC ATC C - Internal Positive Control Reverse oIMR7385 - CCA TGT CTA GAC TGG ACA AGA - Transgene oIMR7386 - CTC CAG GCC ACA TAT GAT TAG - Transgene

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 2.20 - 2.20 2 X SYBR Green Master Mix (Roche) 6.00 1.00 X 6.00 20 uM oIMR7385 0.60 1.00 uM 0.60 20 uM oIMR7386 0.60 1.00 uM 0.60 20 uM oIMR7338 0.30 0.50 uM 0.30 20 uM oIMR7339 0.30 0.50 uM 0.30 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 10 min - 2 94 30 sec - 3 68 1 min - 4 72 45 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 007176 B6.Cg-Tg(Pax8-rtTA2S*M2)1Koes/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.