Stock Number: 008132 Strain Name: STOCK Tg(THY1-Snca)M1mSud/J

Allele: (THY1-Snca)M1mSud Protocol Name: Tg(THY1-SNCA*A53T)M53Sud, F53Sud Method: Melt Curve Analysis Version: 3.0 Created: 04-September-2009 Updated: 01-December -2009

Notes: This assay will NOT distinguish hemizygous from homozygous transgenic animals. Melting curve analysis is done using a Roche Light Cycler 480. Expected Results: Transgene Tm = 86.5°C +/- 1°C Internal positive control Tm = 83°C +/- 1°C Transgene = 500 bp

Gel Image

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Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR8584 - GGC ACC TAG AGG ATC TCG ACT AGT GG - Forward oIMR8585 - GGA CCT CGA CGC TTA GGC TTC AGG - Reverse 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 1.24 - 1.24 20 uM oIMR8584 0.60 1.00 uM 0.60 20 uM oIMR8585 0.60 1.00 uM 0.60 DNA 2.00 - 2.00 10 X High Resolution Melt Master Mix (Roche) 6.00 5.00 X 6.00 25 mM MgCl2 0.96 2.00 mM 0.96 20 ul oIMR8744 0.30 0.50 ul 0.30 20 uM oIMR8745 0.30 0.50 uM 0.30

Cycling  Step # Temp °C Time Note 1 94 10min - 2 94 30 sec - 3 60 1min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold**

This genotyping protocol is used for the following strains:   Stock Number Strain Name 008132 STOCK Tg(THY1-Snca)M1mSud/J 008135 B6.Cg-Tg(THY1-SNCA*A53T)M53Sud/J 008473 B6;SJL-Tg(THY1-SNCA*A30P)M30Sud/J 008474 STOCK Tg(THY1-SNCA*A53T)F53Sud/J 008859 B6.Cg-Tg(THY1-SNCA*A53T)F53Sud/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.