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

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

3. 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 …

4. 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…)

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

6. Nucleic acid quantitation (fluorescence): how?
PicoGreen, OligoGreen, RiboGreen typical procedures
Fluorescent dye
Sample
Excitation
Emission
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

7. Nucleic acid quantitation (fluorescence): how?
Application notes on
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

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

9. 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”

10. 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

11. 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

12. 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

13. 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

14. 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.

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

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