Stock Number: 002749 Strain Name: B6.Cg-Tg(hAGT)2041Sig/J

Allele: Tg(hAGT)2041Sig Protocol Name: Tg(hAGT)2041Sig/J MCA Method: Melt Curve Analysis Version: 1.0 Created: 11-February -2010 Updated: 11-February -2010

Notes: Melting curve analysis is done using a Roche Light Cycler 480. This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene Tm = 90°C +/- 1.5°C Internal positive control Tm = 79°C+/- 1.5°C

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR0585 - TGG TGC TAG TCG CTG CAA AAC TTG ACA CCG - Transgene oIMR0586 - CAG GGA GCA GCC AGT CTT CCA TCC TGT CAC - Transgene 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

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 oIMR0585 0.60 1.00 uM 0.60 20 uM oIMR0586 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 5 min - 2 94 30 sec - 3 59 1 min - 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 002749 B6.Cg-Tg(hAGT)2041Sig/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.