Tuesday, December 1, 2009

Fluorometric Quantitation of dsDNA using Picogreen

Double stranded DNA (dsDNA) is commonly quantified using spectrophotometry at 260 nm without need of additional reagents. However, quantification is limited to 50 ng/mL of dsDNA (0.001 OD) and more typically between 2 and 200 ug/mL in a microplate well sample that is pathlength corrected to 1 cm. Other substances present in dsDNA samples can also absorb at 260 nm including nucleotides, ssDNA, RNA, EDTA, and phenol and skew results (1). Quantification below these concentrations typically requires the addition of reagents, such as fluorophores with selectivity for dsDNA.

Fluorescent dye Hoechst 33258 is inexpensive and more sensitive that spectrophotometry allowing quantification down to 3 ng/mL of dsDNA. Quantitation requires a standard curve and for the best results, top reading solid black plates is recommended. It can bind to ssDNA, however (1,2).

Picogreen® is also a very simple test consisting of TE buffer, Picogreen reagent, a standard curve formed by supplied dsDNA standards and customer supplied samples. Because Picogreen is extremely sensitive, the 20x TE buffer included in the Quant-iT™ PicoGreen dsDNA Assay Kit is certified to be nucleic acid–free and DNase free. The 1x TE working solution is prepared by diluting the supplied 20x buffer with sterile, distilled, DNase free water. Life Technologies (Invitrogen) advertises a dynamic range from 25 pg/mL to 1000 ng/mL (2). Our application note Fluorometric dsDNA Quantification Using PicoGreen® exhibits linearity over the entire dynamic range (3). Average CV's with the Synergy Mx was 2.6% over a 12 standard dynamic range from 0 to 6.4 ng / mL (4).

Some Tips For Using PicoGreen
  • Standard curve dilutions should be prepared in plastic and not glass tubes to avoid the reagent sticking to the glass
  • Picogreen reagent is sensitive to light and wrapping the tube containing the working reagent with tin foil is advisable
  • Picogreen reagent is stable for 3 hours and prepared plates, if covered to reduce evaporation and kept from light, for a least an hour more
  • Aliquots from the tubes are pipetted into the microplate with a total volume of 200 uL per well. Preparing the standards directly in the microplate worsens CV's
  • Samples are best prepared in tubes with the TE buffer but can be prepared in the microplate by adding the Picogreen® working reagent to the wells followed by the sample
  • Detection limits depend on quality of DNA, buffer source, age and storage conditions of Picogreen reagent and the optical quality of the reader and wavelength selections
  • High salts such as 200 nM sodlum chloride and 50 nM of magnesium chloride can depress the signal
  • Because of the extreme sensitivity of this assay, gloves should be worn at all times even when removing microplates from the source box for the plates.
  • Solid black plates such as Corning 3915 provide the lowest background although a few customers report better results for their assays with Corning 3615 low background clear bottomed plates using bottom reading
  • Because of the large dynamic range of the assay, photomultiplier sensitivity setting (gain) should be set high enough so that lower concentrations can be easily differentiated from the blank

References

(1) Held, Paul (2009), [Scientifically Speaking] Quantitation of dsDNA using Hoechst dye 33258, BioTek Instruments, Inc website blog, Winooski, VT

(2) Quant-iT PicoGreen dsDNA Assay kit instruction manual MP07581 "Quant-iT PicoGreen Reagent and Kits".

(3) Held, Paul (2006) Fluorometric dsDNA Quantification Using PicoGreen®, BioTek Instruments, Inc, Winooski, VT

(4) Quigley, Ted (2009), Experimental data

By, BioTek Instruments

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