Protocol for DNA Quantitation Using Hoechst 33258 Dye

1. INTRODUCTION

Quantitation of DNA is a prelude to many practices in Molecular Biology. Common techniques that use DNA, such as sequencing, cDNA synthesis and cloning, RNA transcription, transfection, nucleic acid labeling (e.g. Random prime labeling), etc., all benefit from a defined template concentration. Failure to produce results from these techniques can sometimes be attributed to an incorrect estimate of the DNA template used. DNA concentration is measured by UV absorbance at 260 nm (1A260 = 50 µg/mL) in a 1cm path length cuvette.

The Picofluor™ fluorometer can be used for DNA quantitation along with Hoechst 33258 Dye, a bisbenzimide DNA intercalator that excites in the near UV (350 nm) and emits in the blue region (450 nm). Hoechst 33258 Dye binds to the AT rich regions of double stranded DNA and exhibits enhanced fluorescence under high ionic strength conditions. Sensitivity of the Hoechst 33258 Dye assay is approximately 10 ng/mL. The linear dynamic range extends over 3 orders of magnitude from 10 ng/mL to 1 µg/mL DNA.

2. MATERIALS REQUIRED

• Picofluor™ Laboratory Fluorometer with UV optical configuration
  (P/N 8000-003)
• 10mm x 10mm Methacrylate fluorescence cuvettes (P/N 7000-959).
• FluoReporter Blue Fluorometric ds DNA Quantitation kit (Molecular Probes F-2962)
• Calf Thymus DNA (P/N 3600-941)
• TE buffer for dilution
• 0.45µm filtered water

3. FACTORS TO CONSIDER

3.1 Calf Thymus DNA can often serve as a reference for most plant and animal DNA because it is double-stranded, highly polymerized, and is approximately 58%AT (42%GC). For bacterial DNA, a different standard may be needed because the AT% varies widely depending on species.

3.2 The conformation (supercoiled, relaxed, circular, linear) of plasmid DNA may result in different Hoechst 33258 Dye binding efficiencies. Thus, it is important to select a standard with similar physical characteristics to your sample. The most stable form is linear.

3.3 Hoechst 33258 Dye fluoresces only about half as much when it binds to single-stranded genomic DNA compared to when it binds to double-stranded genomic DNA. In addition, short pieces of single-stranded DNA will not normally cause Hoechst 33258 Dye to fluoresce in proportion to their concentration.

3.4 Buffers commonly used to extract DNA from whole cells have little or no effect on this assay. Low levels of detergent (<0.01%SDS) have little or no effect on this assay.

3.5 Salt concentrations up to 3 M NaCl do not affect this assay. For peak fluorescence, at least 200 mM NaCl is required for purified DNA and 2.0 to 3.0 M for crude samples. In crude samples, higher salt concentrations appear to cause the dissociation of proteins from DNA, allowing the dye molecules to bind easier to DNA.

3.6 RNA does not interfere significantly with the DNA assay because Hoechst 33258 Dye does not normally bind to RNA. Under high salt concentrations, fluorescence from RNA is usually less than 1% of the signal produced from the same concentration of DNA.

4. SOLUTION PREPARATION

4.1 Hoechst 33258 Dye stock dye solution:

NOTE: Hoechst 33258 Dye is a possible carcinogen and possible mutagen. Wear gloves and a mask, and work under a fume hood.

Dilute 100 ul Hoechst 33258 Dye with 100mL TNE buffer (Molecular Probes F-2962). The reagent may be refrigerated if not required immediately. This will be sufficient for 700 samples blanks and standards.

4.2 Calf Thymus DNA Standard: (0.2 A260 = 10 µg/mL). Prepare 4mL of 100-fold diluted (100 ng/mL) stock solution. Dilute with 1X TE to desired concentration as described in Table 1.

Table 1. Protocol for preparing a low range standard curve using Calf Thymus DNA Standard.

5. PROTOCOL FOR GENERATING A STANDARD CURVE

Generating a standard curve verifies the linearity of the assay within a particular concentration range.

NOTE: Accurate pipetting and thorough mixing is critical for reproducible results. However, take extreme care when mixing samples; do not introduce air bubbles. Air bubbles can cause scattering of light leading to inaccurate results. If air bubbles form, hold the upper portion of the cuvette in one hand and gently tap the bottom sides of the cuvette with your other hand to release bubbles.

Set-up the Picofluor™ fluorometer per instructions in the user's manual. Power up the instrument by pressing the [ON/OFF] button. Use the [A/B] button to toggle to the "UV" channel. Press [STD VAL] to program in the concentration of your calibration standard. We suggest calibrating with 100 ng of DNA std. Use the up and down arrows to set the concentration value. Hold down the arrow key to activate faster scrolling. When ready, press the [CAL] button to start the calibration. The Picofluor's screens will lead you through the calibration process.

Measure the fluorescence of the remaining standards to generate a standard curve of fluorescence versus DNA concentration. Figure 1 illustrates the background subtracted fluorescence values (Y- axis) and DNA (ng/mL) (X-axis).

6. SAMPLE ANALYSIS

Dilute the experimental DNA solution in TE to a final volume of 1mL and add 1mL of the Hoechst working solution (prepared in section 4.1) to achieve a final volume of 2.0 mL. You may wish to use two or three different dilution factors for a given sample.

Measure the fluorescence of each sample using the same calibration conditions as used to generate the standard curve (as in section 5). Determine the DNA concentration of each sample from the standard curve generated in section 5.

7. REFERENCES

1. J Histochem Cytochem 24, 24 (1976).
2. Anal. Biochem. 102, 344 (1980).
3. Anal. Biochem. 131, 538 (1983).
4. Anal. Biochem. 191, 31 (1980).
5. Methods Enzymology 58,141 (1979).