Stock Number: 009617 Strain Name: NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A2.1)1Enge/SzJ

Allele: Tg(HLA-A2.1)1Enge Protocol Name: Tg(HLA-A2.1)1Enge Method: Standard PCR Version: 1.1 Created: 31-July -2001 Updated: 20-May -2010

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene = 111 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 oIMR1332 - TTC TCC CTC TCC CAA CCT ATG TAG - Transgene oIMR4311 - CGA CGA CAC TGA TTG GCT TCT - 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 uM oIMR1332 0.45 0.75 uM 0.45 20 uM oIMR4311 0.45 0.75 uM 0.45 20 ul oIMR8744 0.30 0.50 ul 0.30 20 uM oIMR8745 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 20 sec - 3 64 30 sec* *-0.5 C per cycle 4 72 35 sec repeat steps 2-4 for 12 cycles 5 94 20 sec - 6 58 30 sec - 7 72 35 sec repeat steps 5-7 for 25 cycles 8 72 2 min - 9 10 - hold

This genotyping protocol is used for the following strains:   Stock Number Strain Name 003475 C57BL/6-Tg(HLA-A2.1)1Enge/J 004262 NOD.Cg-Prkdcscid Tg(HLA-A2.1)1Enge/Dvs 005512 NOD.B6-Tg(HLA-A2.1)1Enge/DvsJ 009617 NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A2.1)1Enge/SzJ Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.