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: QPCR Version: 4.0 Created: 11-March -2010 Updated: 11-March -2010

Notes: Taqman qPCR protocols are run on an ABI 7500, 7700, 7900 or the Roche LightCycler 480. Use an appropriate instrument-specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

Protocol Primers

Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR1544 - CAC GTG GGC TCC AGC ATT - Internal Positive Control Forward oIMR1770 - TGA CGG GTG TGA CAG CAG TAG - Transgene oIMR1771 - CAG TGG CTG CTG CAA TG - Transgene oIMR3580 - TCA CCA GTC ATT TCT GCC TTT G - Internal Positive Control Reverse TmoIMR0025 6-FAM CCC TGC TCC CTC CAC TGT CTT CTG G TAMRA Tg Probe TmoIMR0105 Cy5 CCA ATG GTC GGG CAC TGC TCA A Black Hole Quencher 2 IC Probe

Number of Reactions

Reaction/Components  Reaction Component Volume (µl) Final Concentration Total Volume (µl) 40 uM oIMR1770 0.04 0.13 uM 0.04 40 uM oIMR1771 0.04 0.13 uM 0.04 40 uM oIMR1544 0.20 0.63 uM 0.20 40 uM oIMR3580 0.20 0.63 uM 0.20 5 uM TmoIMR0025 0.38 0.15 uM 0.38 5 uM TmoIMR0105 0.65 0.25 uM 0.65 DNA 5.00 0.00 5.00 2 X PerfeCTa qPCR SuperMix 6.25 0.00 X 6.25

Cycling  Step # Temp °C Time Note 1 50 2 min - 2 95 10 min - 3 95 15 sec - 4 60 1 min repeat steps 3-4 for 40 cycles

This genotyping protocol is used for the following strains:   Stock Number Strain Name 004479 B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J 010710 FVB;129S6-Sncatm1Nbm Tg(SNCA)1Nbm/J 010788 FVB;129S6-Sncatm1Nbm Tg(SNCA*A30P)1Nbm Tg(SNCA*A30P)2Nbm/J 010799 FVB;129S6-Sncatm1Nbm Tg(SNCA*A53T)1Nbm Tg(SNCA*A53T)2Nbm/J 017678 FVB;129-Pink1tm1Aub Tg(Prnp-SNCA*A53T)AAub/J Genotyping resources and troubleshooting This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.