Stock Number: 004696 Strain Name: NOD.Cg-Tg(TcrLCMV)327Sdz/DvsJ

Allele: Tg(TcrLCMV)327Sdz Protocol Name: Tg(TcrLCMV)327Sdz Method: Standard PCR Version: 1.1 Created: 09-July -2008 Updated: 18-April -2013

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene = 400 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 oIMR0244 - CAT GGA GGC TGC AGT CAC CC - Transgene oIMR0245 - GTT TGT TTG CGA GCT CTG TTT TGA TGG CTC - Transgene 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 3.66 - 3.66 10 X AB PCR BufferII 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 ul oIMR8744 0.45 0.75 ul 0.45 20 uM oIMR8745 0.45 0.75 uM 0.45 20 uM oIMR0244 0.30 0.50 uM 0.30 20 uM oIMR0245 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 94 3 min - 2 94 30 sec - 3 61 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 004257 NOD.Cg-Prkdcscid Tg(TcrLCMV)327Sdz/Dvs 004694 B6;D2-Tg(TcrLCMV)327Sdz/JDvsJ 004696 NOD.Cg-Tg(TcrLCMV)327Sdz/DvsJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.