Fluorescence nucleic acid quantitation DNA oligonucleotides RNA Nucleic

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Fluorescence: nucleic acid quantitation (DNA, oligonucleotides, RNA…)

Fluorescence: nucleic acid quantitation (DNA, oligonucleotides, RNA…)

Nucleic acid quantitation (fluorescence) • • Why? - Applications How? – Principle Example in

Nucleic acid quantitation (fluorescence) • • Why? - Applications How? – Principle Example in KC 4 What do you need to make this work?

Nucleic acid quantitation (fluorescence): why? • Extremely large range of applications • Used extensively

Nucleic acid quantitation (fluorescence): why? • Extremely large range of applications • Used extensively in a lot of labs • When the limit of detection of the 260 nm measurement (250 ng/ml of ds. DNA) is not enough • DNA sequencing after PCR amplification Ø Critical to know DNA concentration prior to sequencing • Preparation and optimization of a PCR amplification Ø Critical to know the amount of primers used in the PCR reaction • …

Nucleic acid quantitation (fluorescence): why? • More sensitive than 260 nm OD measurement Ø

Nucleic acid quantitation (fluorescence): why? • More sensitive than 260 nm OD measurement Ø Allows to go in the pg/well range Ø Versus ng/well to µg/well in absorbance mode • More molecule-specific. Requires specific dyes, for example: Ø Pico. Green for double-stranded DNA (ds. DNA) Ø DABA for single-stranded hydrolized DNA Ø Oligo. Green for single-stranded oligos (ss. DNA with< 25 nucleotides) Ø Ribo. Green for RNA Ø Measurement of OD at 260 nm does not make any difference between these different molecules (ds. DNA, ss. DNA, RNA…)

Nucleic acid quantitation (fluorescence) • • Why? - Applications How? – Principle What do

Nucleic acid quantitation (fluorescence) • • Why? - Applications How? – Principle What do you need to make this work? Example in KC 4

Nucleic acid quantitation (fluorescence): how? Pico. Green, Oligo. Green, Ribo. Green typical procedures Excitation

Nucleic acid quantitation (fluorescence): how? Pico. Green, Oligo. Green, Ribo. Green typical procedures Excitation Sample Emission Fluorescent dye • Typically 100µl of sample is used • Typically 100µl of dye solution is added • Fluorescence can be measured after a 5 minutes incubation at room temperature (read with fluorescein filter set) • Extremely simple procedures

Nucleic acid quantitation (fluorescence): how? Application notes on www. biotek. com • • •

Nucleic acid quantitation (fluorescence): how? Application notes on www. biotek. com • • • Quantitation of ss. DNA using Oli. Green Fluorescent Stain Quantitation of RNA with Ribo. Green Stain Using the FL 600 Fluorometric Quantitation of ds. DNA Fluorometric Quantitation of Hydrolyzed DNA Increasing the Range of DNA Quantitation when using Hoechst Dye 33258

Nucleic acid quantitation (fluorescence) • • Why? - Applications How? – Principle Example in

Nucleic acid quantitation (fluorescence) • • Why? - Applications How? – Principle Example in KC 4 What do you need to make this work?

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green typical procedures Reading parameters: • End-point • Fluorescence • Excitation 485/20 Emission 528/20 • Optics position “Top” • Click on “options”

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green typical procedures Reading parameters: • Set “Delay before sampling” to 10 ms (speeds-up the read) • Check “Automatic sensitivity adjustment” and select “Scale to High wells” • High well: enter the position of a high standard well • High value: 70, 000 • Staring sensitivity: 25 • Click OK

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green typical procedures Plate Layout: • In KC 4 go to “Layout” • Well Type: select “Standard” • Click on “…” to define the • concentrations of the standards Click “OK” and define the position of each standard on the plate map • Add “Blank” wells if any • Add sample wells

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green typical procedures Typical plate layout: • 6 standards in duplicate • Samples in duplicate • 2 blank wells

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green typical procedures Standard curve: • Go to “Curve” in KC 4 • Select the appropriate curve fit (see kit information). If information not available, try “Linear Regression” and see results

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green

Nucleic acid quantitation (fluorescence): example in KC 4 Pico. Green, Oligo. Green, Ribo. Green typical procedures Results: • KC 4 automatically generates a standard curve • The concentrations of the unknown samples are automatically calculated from the curve.

Nucleic acid quantitation (fluorescence) • • Why? - Applications How? – Principle Example in

Nucleic acid quantitation (fluorescence) • • Why? - Applications How? – Principle Example in KC 4 What do you need to make this work?

Nucleic acid quantitation (fluorescence): tips • Make sure you use low-background solid black microplates

Nucleic acid quantitation (fluorescence): tips • Make sure you use low-background solid black microplates (i. e. Corning 3915, Greiner 655076…) • Make sure you have the right filters before going to the demo