BioMicroCenter:BioMark

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Contents

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.

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