Stock Number: 005443 Strain Name: CBy.PL(B6)-Thy1a/ScrJ

Allele: Tg(TcraCl4,TcrbCl4)1Shrm Protocol Name: Tg(TcraCl4,TcrbCl4)1Shrm Method: Separated PCR Version: 1.1 Created: 16-June -2008 Updated: 24-September-2009

Expected Results: Transgene (Tcra) = 200 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 oIMR4123 - TGA TTT AAC AGA GAG TTT AG - - oIMR4124 - CCT CGT CAG CCT GTT GTC CAA TCC TTC TGG - - 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

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 oIMR4123 0.60 1.00 uM 0.60 20 uM oIMR4124 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 58 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold

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 oIMR7338 0.60 1.00 uM 0.60 20 uM oIMR7339 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 This genotyping protocol is used for the following strains:   Stock Number Strain Name 005307 CBy.Cg-Thy1a Tg(TcraCl4,TcrbCl4)1Shrm/ShrmJ 005308 B10.Cg-H2d Tg(TcraCl4,TcrbCl4)1Shrm/ShrmJ 005443 CBy.PL(B6)-Thy1a/ScrJ 005686 NOD.Cg-Thy1a Tg(TcraCl4,TcrbCl4)1Shrm/ShrmJ 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 58 1 min - 4 72 1 repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold