Stock Number: 005870 Strain Name: NOD/ShiLt(Cg)-Tg(Ins2-GAD2)2Lt/J

Allele: Tg(Ins2-GAD2)2Lt Protocol Name: Tg(Ins2-GAD2) Method: Standard PCR Version: 1.1 Created: 21-June -2008 Updated: 24-September-2009

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 oIMR6019 - AGC ACT TTC TGC AGA CCT AGC AC - Transgene oIMR6020 - GGC TTC TCG GCG TCT CCG TAG A - 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 3.36 - 3.36 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 0.30 0.50 ul 0.30 20 uM oIMR8745 0.30 0.50 uM 0.30 20 uM oIMR6019 0.60 1.00 uM 0.60 20 uM oIMR6020 0.60 1.00 uM 0.60 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 60 30 sec - 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 003074 NOD/ShiLt-Tg(Ins2-GAD2)1Lt/LtJ 003843 NOD.Cg-Prkdcscid Tg(Ins2-GAD2)1Lt/LtJ 003844 NOD.Cg-Prkdcscid Tg(Ins2-GAD2)2Lt/LtJ 005870 NOD/ShiLt(Cg)-Tg(Ins2-GAD2)2Lt/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.