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

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

Nucleic acid quantitation (fluorescence) • Why? - Applications • How? – Principle • Example in KC4 • What do you need to make this work?

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

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

Nucleic acid quantitation (fluorescence) • Why? - Applications • How? – Principle • What do you need to make this work? • Example in KC4

Excitation Emission Fluorescent dye Sample Nucleic acid quantitation (fluorescence): how? PicoGreen, OligoGreen, RiboGreen typical procedures • 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 • Quantitation of ssDNA using OliGreen Fluorescent Stain • Quantitation of RNA with RiboGreen Stain Using the FL600 • Fluorometric Quantitation of dsDNA • Fluorometric Quantitation of Hydrolyzed DNA • Increasing the Range of DNA Quantitation when using Hoechst Dye 33258

Nucleic acid quantitation (fluorescence): example in KC4 PicoGreen, OligoGreen, RiboGreen 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 KC4 PicoGreen, OligoGreen, RiboGreen 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 KC4 PicoGreen, OligoGreen, RiboGreen typical procedures • Plate Layout: • In KC4 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 KC4 PicoGreen, OligoGreen, RiboGreen typical procedures • Typical plate layout: • 6 standards in duplicate • Samples in duplicate • 2 blank wells

Nucleic acid quantitation (fluorescence): example in KC4 PicoGreen, OligoGreen, RiboGreen typical procedures • Standard curve: • Go to “Curve” in KC4 • Select the appropriate curve fit (see kit information). If information not available, try “Linear Regression” and see results

Nucleic acid quantitation (fluorescence): example in KC4 PicoGreen, OligoGreen, RiboGreen typical procedures • Results: • KC4 automatically generates a standard curve • The concentrations of the unknown samples are automatically calculated from the curve.

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

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