I am having 40 ng/ul of plasmid DNA and would like to concentrate it to get at least 10 to 20 times concentrated solution 400 ng-800 ng/ ul.
Centrifuge by AMICON ?
Freeze dry?
Precipitation by sodium acetate?
I am not sure which one is the best way to maintain the quality of plasmid DNA.
Sachin S. Surwase
1. You can use SpeedVac (centrifugation) to concentrate your DNA if your DNA solution is pure enough. If your DNA is not pure, the contaminants in DNA solution can be enriched using this method, and influence downstream experiments.
2. Another method is through precipitation. Here is a protocol provided by Qiagen. Using this method, you might lost some DNA.
< https://www.qiagen.com/us/resources/faq?id=5d591b8b-968a-4a17-849f-9d0f719b40af&lang=en >
- Add 1/10 volume of 3 M Na-Acetate pH 5.2, and 2 to 2.5 volumes of ice-cold 100% ethanol to the DNA sample
- Mix, and store at -20°C for at least 1 hour to precipitate the DNA
- Recover the precipitated DNA by centrifugation at full speed in a microcentrifuge for 15-20 minutes
- Pour off the ethanol and wash the pellet twice with room-temperature 70% ethanol
- Allow the DNA pellet to air-dry
- resuspend the DNA in a suitable volume of sterile TE buffer or distilled water
I agree with the suggestion to use a speedvac if one is available. Other good options are to use a DNA purification column or DNA cleanup beads (Ampure) which would be faster and easier than a precipitation step and likely retain more of your sample. If you don't have access to those, then precipitation is your best option.
Sachin S. Surwase
In my personal opinion, Freeze-drying works well for concentrating nucleic acids as well as proteins. But Yuan-Yeu Yau said, concentrating the sample through speedvac and freeze drying can increase the salt concentration as well. Take care of that before proceeding.
Precipitation with sodium acetate before speedvac or freeze drying would be an ideal way to go forward
Cheers!
Thank you for your suggestions! Ashis Kumar Laura Leighton Yuan-Yeu Yau
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