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Application Data

- Determining quantity of Double-stranded DNA with PicoGreen® -

Feature

Quantitative estimation without influence of adulterated material in sample such as RNA, single-stranded DNA, protein, and so on.

Intended purpose

This protocol is widely used in various measurement such as measurement of DNA template quantity for High-throughput capillary DNA sequencer, measurement of template in DNA amplifying PCR method and amplified DNA quantity. DNA quantitative method with PicoGreen® enables to measure dsDNA specifically and is suitable to DNA sequence and quantitive measurement of template with high-throughput.

Detection limit : 250pg/ml Range of linearity : MTP-650FA FL microplate reader Reagent : Molecular Probes PicoGreen® dsDNA quantitation reagent '200-2000 assays' DNA solution with known concentration for working curve TE buffer : TE buffer: for diluting reagent and sample

Analysis procedure
1. Making up Reagent and Sample
	a) Making up PicoGreen® solution
		Dilute stock solution with TE 200 times making total quantity as much as necessary. (100µL/well)       ∴ Keep the made solution from light by shielding with tinfoil or something.
	b) Making up Std DNA for working curve
		Make up standard DNA by Diluting DNA solution of known concentration with TE.
	c) Diluting of sample DNA
		Dilute sample DNA according to need.
2. Measurement
Dispense 100µL of PicoGreen® solution to each well of microplate. Add 100µL of standard DNA and sample DNA and mix with pipette. Incubate the plate in room temperature for five minutes. Fluorometric measurement.

Condition of analysis
Meas. Method : End point Flash times : 20 MODE1 : FL intensity MODE2 : 1Ex-1Em Ex : 490nm Em : 530nm SENS : ×1 Direction : Vertical TEMP : OFF Mode : END FL focus : 4		Data is provided from HHT BMC.

* PicoGreen® is registered brand of Molecular Probes, Inc.

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