Isopropanol DNA Precipitation - Best for DNA Concentration
Lynn Doran
Abstract
Protocol to precipitate extracted DNA from an aqueous solution to increase concentration or resolubilize in a different storage buffer.
*Use isopropanol DNA precipitation if your DNA is suspended in a very large volume, if your DNA concentration is low, or you are trying to concentrate large molecular weight DNA fragments and remove smaller fragments.
*Use ethanol DNA precipitation if you are trying to remove salt contamination or precipitate small DNA fragments.
Additional Resources:
Steps
Transfer desired amount of aqueous DNA solution to a fresh microtube. (70 ul)
Add 3.0 M pH 5.2 sodium acetate to the DNA solution to result in a final concentration of 0.3 M sodium acetate. (7 ul)
Add 0.6–0.7 volume of isopropanol at room temperature and mix well. (50 ul)
Point caps of tubes out to help identify pellet location below cap. Centrifuge the sample immediately at 10,000–15,000 x g for 20–30 min in a microcentrifuge tube at 4°C.
Identify the pelleted DNA. Carefully decant the supernatant fluid into a fresh labeled tube without losing the pellet.
Wash the DNA pellet by adding 500 ul or more (depending on the size of the preparation) of room-temperature 70% ethanol. Flick tube and invert several times until pellet is removed from the side of the tube and all sides can be washed.
Centrifuge the sample at 10,000–15,000 x g for 20–30 min at 4°C.
Visually identify the pellet. Pour out ethanol. Tap tube gently on paper towel, being sure to keep track of the pellet.
Optional: For high quality DNA applications requiring very clean DNA extractions, repeat steps 6-8. More ethanol washes will increase quality but will negatively affect recovery.
After last ethanol wash, use a pipette to remove any ethanol that remains after decanting.
Allow samples to air dry at room temperature for 10-20 min. When pellet turns from white to clear the pellet is dry. Do not over dry.
Resuspend the DNA pellet in Tris, TE (pH 8.0), or molecular grade water. Incubate in 4°C overnight for more complete resuspension. For long term storage, store at -20°C.
