The plasmid i am isolating is a low copy number plasmid. I am supplementing the broth culture with appropriate antibiotics. Even then the plasmid quantity i am getting is not satisfactory. The mini spin columns i am using are quite old and what i can conclude is that the silica membrane in the columns is not binding the DNA properly and the bulk isolate it getting washed away. I would like to know as how to enhance the efficiency of mini spin columns for plasmid prep.
Any other suggestions from your side would be highly appreciated
Regards
If you look at the columns under the microscope you will probably find the silica membrane has cracked so your DNA is just flowing through the cracks without binding. Get new columns. Once you have applied your lysate to the column, let the column stand for 5-10 minutes before centrifuging to allow the silica to equilibrate. After performing the ethanol wash leave the column for 10 minutes with the lid off to allow for remaining ethanol to evaporate. When eluting the plasmid incubate the column for 1-2 mins before centrifuging and passing the eluted plasmid solution through the column 2-3 times. You can also do several minipreps in parallel and elute using the plasmid containing solution from the previous column. Alternatively combine you plasmid solutions into a large volume and precipitate with isopropanol (60% total volume on ice), centrifuge, wash with 70% ethanol and then resuspend in the desired volume.
#Robert Adolf Brinzer
Thank you for your suggestions.
I would like to know is there any method by which the silica membrane is reactivated. I read somewhere that wetting the column with aqueous solution preferably by a nucleophile compound like ethanol, sorbitol or water helps in reactivating the silica surface thus enhances binding. is that the case?
Ethanol is mostly to remove any water that has condensed in the membrane. It will not help against cracks in the silica resin which is non-soluble.
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