DNA Sequencing and Fragment Analysis

Archive for July, 2012

Protocol: Alternative Method for Sodium Acetate to Ethanol Precipitate DNA

Ethanol with sodium acetate is used as a method for precipitating DNA in solution. It is preferred over simple drying in order to concentrate DNA. Drying concentrates salts and contaminants in addition to the DNA. Ethanol precipitation allows contaminants to be removed along with the excess liquid while the DNA forms a solid pellet on the bottom of the tube. It is a good method. Isopropanol is also used as a replacement for ethanol. It requires less volume. However, we recommend ethanol, as it seems to clean the DNA more efficiently.

Sodium acetate is a salt. The salt is a necessary ingredient because it acts as a carrier for the DNA. Without sodium acetate, the majority of the DNA would remain in solution. It would be lost along with the other contaminants.

The following method works well for final purification of PCR fragments and plasmid preparations with a volume of 20 to 25 ul. Glycogen replaces sodium acetate as the DNA carrier.

Things You Need

95 % Ethanol

70 % Ethanol

Glycogen (20 mg/ ml)

Microcentrifuge tubes (1.5 ml)

Microcentrifuge

Hot plate with tube block

Protocol

Step 1: Add 1.5 ul glycogen to DNA in solution. It is important to add the glycogen before adding ethanol because ethanol will cause glycogen to precipitate.

Step 2: Add 80 ul 95 % ethanol.

Step 3: Vortex to mix components.

Step 4: Allow DNA and glycogen to precipitate for a minimum of 15 minutes.

Step 5: Centrifuge for 15 minutes to pellet DNA.

Step 6: Remove excess ethanol by decanting or using a pipette.

Step 7: Add 200 ul 70 % ethanol

Step 8: Mix gently by inverting the tube several times.

Step 9: Centrifuge for 15 minutes.

Step 10: Remove excess ethanol by decanting or using a pipette.

Step 11: Dry in heat block to remove remaining ethanol.

Step 12: Re-suspend in 10 ul purified water.

Step 13: Determine concentration.

Variations to the Protocol

Temperature and time are 2 variables that effect the precipitation of DNA. Often precipitation is performed at 4O C to improve yields. Time is also a factor. Precipitating for longer periods could improve yields. However, using lower temperatures and longer precipitation times could drop certain contaminants out of solution. Some caution should be used when changing both variables.

Once the DNA pellet is dry, it could be re-suspended in water or a buffer of choice. Water has the advantage of adding no additional salts initially removed during the precipitation. The disadvantage of water is that DNA is more easily subject to degradation. Water is a reasonable diluent for short-term applications or Sanger sequencing. Some sequencing facilities recommend water for the final aliquot in preparation of samples. TRIS and TRIS with EDTA are also used for long-term storage of DNA samples. Buffers containing EDTA are not recommended for samples submitted for Sanger sequencing and PCR because EDTA interferes with the MgCl2, a necessary component for amplification.

Please submit any additional suggestions or questions related to the glycogen protocol for ethanol precipitation of DNA.

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