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Tips from the Bench: Nucleic Acid Labeling Tip

Post-labeling Purification of Nucleic Acids with the Label IT® Reagents

Labeled nucleic acids need to be purified to remove the Label IT® dye and other reaction components that might affect downstream steps. Purification can be accomplished by ethanol precipitation, silica-based column purification, or gel filtration using G50 microspin purification columns. If the purified labeled sample is to be quantified by measuring absorbance at 260 nm (A260), the sample must be purified by either ethanol precipitation or silica-based columns. Use of G50 microspin columns produces purified samples with erroneously high A260 readings. It is generally acceptable to assume 100% recovery of the labeled nucleic acid following G50 microspin column purification.

Purify Labeled DNA or RNA Using Ethanol Precipitation

Ethanol precipitation is a highly effective, low cost method for concentrating and purifiying nucleic acids. Ethanol precipitation is recommended for the purification of labeled nucleic acids in many of the Label IT® kits. When performed properly, nucleic acid recovery by ethanol precipitation is ≥70%. Follow the DNA- or RNA-specific ethanol precipitation protocols and always use DNAse-, RNase-free reagents to prevent degradation of your nucleic acid samples.

Ethanol precipitation protocol for samples containing ≥1 µg nucleic acid

  1. Add enough water to your sample to bring the final volume to 200 µl and mix thoroughly.
  2. For each type of nucleic acid, add the following:

    - 20 µl (0.1X vol.) 5M NaCl
    - 400 µl (2X vol.) ice-cold 100% ethanol

    - 20 µl (0.1X vol.) 5M NaCl
    - 500 µl (2.5X vol.) ice-cold 100% ethanol

  3. Cap the tube and mix completely by inversion.
  4. Incubate the tube at -20ºC or colder for ≥30 minutes.
  5. Centrifuge at 4ºC in a microcentrifuge at maximum speed for ≥10 minutes.
    Note: Consistently orient the tube in the microcentrifuge so that you know where the nucleic acid pellet will be located after centrifugation. It is often difficult to see pellets from small amounts of nucleic acid.
  6. Gently remove the supernatant using a micropipet without disturbing the nucleic acid pellet.
  7. Add 500 µl of room temperature 70% ethanol.
  8. Centrifuge at 4ºC in a microcentrifuge at maximum speed for ≥10 minutes.
  9. Gently and completely remove the supernatant using a micropipet without disturbing the nucleic acid pellet.
  10. Recentrifuge the microfuge tube for 5 seconds, and gently remove any remaining ethanol using a micropipet.
  11. Immediately resuspend the nucleic acid pellet in a suitable buffer for the downstream application (for example, TE buffer).

Use Glycogen as Carrier to Help Precipitate Small Fragments or Limiting Amounts of Nucleic Acid

To enhance recovery from samples containing limiting amounts (<1 µg) of nucleic acid or small nucleic acids (≤100 nucleotides or base pairs), inert carriers such as glycogen can be added during ethanol precipitation. Follow the above protocol and add glycogen to a final concentration of 50-150 µg/ml of the nucleic acid sample before adding NaCl and ethanol. The addition of glycogen will also make the precipitated pellet easier to visualize in the bottom of the microfuge tube.

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