Stock Number: 008099 Strain Name: FVB-Tg(KRT14-rtTA)F42Efu/J

Allele: Tg(KRT14-rtTA)F42Efu Protocol Name: Tg(KRT14-rtTA)F42Efu Method: Standard PCR Version: 1.1 Created: 11-July -2008 Updated: 16-November -2009

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene = ~500 bp Internal positive control = 200 bp

Gel Information: Separated by gel electrophoresis on a 1.5% agarose gel.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR7862 - CAC GAT ACA CCT GAC TAG CTG GGT G - Transgene Forward oIMR7863 - CAT CAC CCA CAG GCT AGC GCC AAC T - 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 3.82 - 3.82 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 oIMR7862 0.90 1.50 uM 0.90 20 uM oIMR7863 0.90 1.50 uM 0.90 20 oIMR8744 0.60 1.00 0.60 20 uM oIMR8745 0.60 1.00 uM 0.60 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 68 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 008099 FVB-Tg(KRT14-rtTA)F42Efu/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.