BioMicroCenter:BioMark: Difference between revisions
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* [[Media:Fluidigm_RT-_PCR_Analysis_v2.1_User_Guide_.pdf|Fluidigm RT- PCR Analysis Softwarev2.1 User Guide]] | * [[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_RT_PCR_Analysis_brief_manual_07302010.doc|Fluidigm_RT_PCR Analysis brief Protocol]] | ||
* [[Media: | * [[Media:Fluidigm_96.96_RT-_PCR_Quick_Reference_.pdf | Fluidigm 96.96 RT- PCR Quick Reference ]] |
Latest revision as of 13:07, 23 August 2010
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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.