Training Session Notes:
- Highly multiplex PCR.
TaqMan Intercalating Dye (EvoGreen) Roche UPL
Genotype w/ Taqman - recommend 50 copies per cell. 50ng. 30ng/ul starting material
Single Cell (requires Taqman)
1ng - 250ng is standard input Cell direct Kit from Invitrogen for single cells Use 2-3 lines per sample as replicates.
- Specific target preamplification
48/96 Asssys mix (@180nM) cDNA sample preamp master mix
14 (reg) / 18 (1 cell) cycles of PCR Dilute 1:5
- prep assays + samples
2x Master mix 2.50ul / sample - NEEDS ROX - ABI universal master mix Loading reagent 0.25ul / sample (detergent to reduce surface tension) cDNA 2.25ul pre amp
20x primer probe 2.5ul (TaqMan) Loading Reagent2 2.5ul detergent
- prime chip
- pipette samples + assays
- load / mix chip
- Data Analysis
Plots are heatmaps and RT curves similar plots for deltaCT and DDCT
- Dynamic PCR: 48x48 or 96x96 - relative quantification
- Digital PCR: 12x765 or 48x770 - rigorous quantification
Big raised areas on array are the Air Tanks
48.48 - 9nL of sample 1nL of assay 96.96 - 6.7nL of sample 0.7nL of assay mix by diffusion
Manual and Protocols
Manual and Protocol from Fluidigm are available.