Stock Number: 003741 Strain Name: B6D2-Tg(Prnp-MAPT)43Vle/J

Allele: Tg(Prnp-MAPT)43Vle Protocol Name: Tg(Prnp-MAPT)43Vle Method: Standard PCR Version: 1.1 Created: 27-July -2005 Updated: 24-September-2009

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene = ~150 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 oIMR1369 - GGG AGT TCG AAG TGA TGG AA - Transgene oIMR1370 - TCT CCA ATG CCT GCT TCT TC - 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 3.39 - 3.39 10 X PCR Buffer II (ABI) 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 oIMR1369 0.60 1.00 uM 0.60 20 uM oIMR1370 0.60 1.00 uM 0.60 20 oIMR8744 0.30 0.50 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 0.03 0.01 U/ul 0.03 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 003741 B6D2-Tg(Prnp-MAPT)43Vle/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.