Pico488 DNA quantification kit manual

The Pico488 DNA quantification kit is designed to measure double-stranded DNA (dsDNA) concentration if the concentration is low and cannot be measured spectrophotometrically at 260 nm. As Pico488 selectively binds to dsDNA, the results are independent of nucleotides, single-stranded DNA, RNA, proteins and other impurities in the sample, if present.

The range of linearity for DNA concentration measurements is 1 pg/μL – 5 ng/μL.

Kit components

The specified components are suitable for 200 measurements with a sample volume of 2 mL.

Component Characteristics Quantity
Pico488 dye, 1 mL Solution in DMSO 1 ea
20× TE Buffer, 25 mL 200 mM Tris HCl, 20 mM EDTA, pH 7.5 1 ea
E. coli genomic DNA standard, 1 mL 100 ng/µL 1 ea

Store at temperature below 4°С. Do not freeze! Shelf life 1 year.

Protocol

1. Buffer preparation.

To get needed volume of buffer, dilute the stock buffer 20-fold. For example, if you need to perform 10 measurements, dilute 1 mL of the buffer with 19 mL of ddH2O in order to get 20 mL of 1× buffer.

2. Pico488 aqueous solution preparation.

Thaw the content of Pico488 dye vial and dilute needed amount of the Pico488 dye solution 200-fold with the 1× buffer. For example, to perform 20 measurements add 100 µL of Pico488 dye solution to 19.9 mL of 1× buffer. Mix and use in the course of 3 hours max.

3. DNA solutions preparation.

Prepare a E.Coli DNA standard stock solution at a concentration of 2 ng/µL: 30 µL of the DNA standard should be mixed with 1.47 mL of 1× buffer. Prepare solutions at the following concentrations using the stock solution: 1 ng/µL, 100 pg/µL, 10 pg/µL, 1 pg/µL (see the table below for the components). A narrower range of concentrations may be used if needed.

1× buffer volume, µL DNA standard stock solution volume, µL Pico488 aqueous solution volume, µL Final concentration of DNA solutions
0 1000 1000 1 ng/µL
900 100 1000 100 pg/µL
990 10 1000 10 pg/µL
999 1 1000 1 pg/µL
1000 0 1000 0

4. Sample solution preparation.

Dissolve your DNA sample in 1× buffer to get 1 mL of the solution (you may take any DNA amount you want). Add 1 mL of Pico488 aqueous solution. Mix and incubate for 5 min.

5. Fluorescence measurements.

Measure fluorescence from standard and sample DNA solutions in a fluorimeter. Absorption wavelength is 503 nm, emission wavelength is 525 nm.

6. Calculation of concentration.

Plot fluorescence vs concentration using any software. Obtain linear equation of fluorescence (FL) vs concentration (C) dependence:
FL= A × C + B.

To calculate DNA concentration in the sample solution:
Csample = (FLsample – B)/A, where FLsample is sample solution fluorescence.

To calculate sample DNA concentration:
Сinit = 2000 × Csample/V, where V is the volume of your initial DNA sample, µL.

Example:

Related kits

Pico488 DNA quantification kit

A kit for the quantification of dsDNA using Pico488 fluorescent dye.
Show pricing
Cat. # Quantity Price Lead time
1102-200 200 assays $350.00 in stock

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