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

Allele: Tg(hAGT)2041Sig Protocol Name: Tg(hAGT)2041Sig Method: Standard PCR Version: 2.1 Created: 07-December -2006 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). This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene = 539 bp Internal positive control = 324 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 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 3.60 - 3.60 10 X AB PCR BufferII 0.96 0.80 X 0.96 25 mM MgCl2 0.96 2.00 mM 0.96 2.5 mM dNTP 0.96 0.20 mM 0.96 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 5 mM DNA Loading Dye 1.66 0.69 mM 1.66 5 U/ul Taq DNA Polymerase + TaqStart AB (1:1) 0.06 0.01 U/ul 0.06 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 94 1.5 min - 2 94 30 sec - 3 60 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 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.