Stock Number: 008202 Strain Name: FVB/N-Tg(NPHS2-rtTA2*M2)1Jbk/J

Allele: Tg(NPHS2-rtTA2*M2)1Jbk Protocol Name: Tg(NPHS2-rtTA2*M2)1Jbk Method: Standard PCR Version: 1.1 Created: 11-July -2008 Updated: 24-September-2009

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene = 196 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 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 oIMR8111 - GAA CAA CGC CAA GTC ATT CCG - Transgene Forward oIMR8112 - TAC GCA GCC CAG TGT AAA GTG G - Transgene Reverse

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) ddH2O 3.36 - 3.36 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 oIMR8111 0.60 1.00 uM 0.60 20 uM oIMR8112 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 0.06 0.03 U/ul 0.06 DNA 2.00 - 2.00

Cycling  Step # Temp °C Time Note 1 95 5 min - 2 94 30 sec - 3 58 30 sec - 4 72 40 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 008202 FVB/N-Tg(NPHS2-rtTA2*M2)1Jbk/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.