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.
The specified components are suitable for 200 measurements with a sample volume of 2 mL.
|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.
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|
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.
Сinit = 2000 × Csample/V, where V is the volume of your initial DNA sample, µL.
|1102-200||200 assays||$350.00||Auf Lager|
|B1102||200 assays||$395.00||Auf Lager|