Stock Number: 004479 Strain Name: B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J

Allele: Tg(Prnp-SNCA*A53T)83Vle Protocol Name: Tg(Prnp-SNCA*A53T)83Vle Method: Standard PCR Version: 1.1 Created: 07-May -2004 Updated: 24-September-2009

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Expected Results: Transgene = 248 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 oIMR1772 - TGT AGG CTC CAA AAC CAA GG - Transgene oIMR3560 - TGT CAG GAT CCA CAG GCA TA - Transgene 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 3.36 - 3.36 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 oIMR1772 0.30 0.50 uM 0.30 20 uM oIMR3560 0.30 0.50 uM 0.30 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

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 004479 B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.