BioMicroCenter:BioMark

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Training Session Notes:

• Highly multiplex PCR.

Assays

TaqMan Intercalating Dye (EvoGreen) Roche UPL

Genotype w/ Taqman - recommend 50 copies per cell. 50ng. 30ng/ul starting material

miRNA

Single Cell (requires Taqman)

WorkFlow

• Input

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
• RT-PCR
• Data Analysis

  Plots are heatmaps and RT curves
  similar plots for deltaCT and DDCT

Chip Types

• 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.

• Fluidigm RT- PCR Analysis Softwarev2.1 User Guide
• Fluidigm_RT_PCR Analysis brief Protocol
• Fluidigm 96.96 RT- PCR Quick Reference