BioMicroCenter:BioMark

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
1ng - 250ng is standard input Cell direct Kit from Invitrogen for single cells Use 2-3 lines per sample as replicates.
 * Input


 * 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 ]]