Stock Number: 009377 Strain Name: NOD.Cg-Rag1tm1Mom Tg(TcraBDC12-4.1)10Jos Tg(TcrbBDC12-4.1)82Gse/J

Allele: Tg(TcrbBDC12-4.1)82Gse Protocol Name: Tg(TcrbBDC12-4.1)82Gse Method: Standard PCR Version: 1.1 Created: 25-June -2008 Updated: 24-September-2009

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene = 700 bp Internal positive control = 200 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 oIMR6265 - GTC TTG TTT CAG ACC CCA CAG - Transgene oIMR6500 - CTA CCT TGT CCT GGC TTG CG - - 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 3.96 - 3.96 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 uM oIMR6265 0.60 1.00 uM 0.60 20 uM oIMR6500 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 54 30 sec - 4 72 30 sec repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold

Reaction/Components  None found. This genotyping protocol is used for the following strains:   Stock Number Strain Name 006304 NOD.FVB-Tg(TcrbBDC12-4.1)82Gse/GseJ 009377 NOD.Cg-Rag1tm1Mom Tg(TcraBDC12-4.1)10Jos Tg(TcrbBDC12-4.1)82Gse/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.

Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 54 30 sec - 4 72 30 sec repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold