Editing BioMicroCenter:BioMark

{{BioMicroCenter}}

== 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.

* [[Media:Fluidigm_RT-_PCR_Analysis_v2.1_User_Guide_.pdf|Fluidigm RT- PCR Analysis  Softwarev2.1 User Guide]]  
* [[Media:Fluidigm_RT_PCR_Analysis_brief_manual_07302010.doc|Fluidigm_RT_PCR Analysis brief Protocol]]  
* [[Media:Fluidigm_96.96_RT-_PCR_Quick_Reference_.pdf | Fluidigm  96.96 RT- PCR Quick Reference ]]