Principle of silica membrane binding of nucleic acid
Posted 09 March 2013 - 10:10 AM
Posted 09 March 2013 - 01:14 PM
Posted 09 March 2013 - 07:38 PM
Posted 10 March 2013 - 12:14 AM
Posted 10 March 2013 - 08:27 AM
And why ethanol elution can not remove DNA or RNA?
Bob, could you please give me a detailed explanation from the lysis of bacteria or virus with alkaline buffer or enzyme, then binding the lysis solution to the column, and elution with several buffers and ethanol, finally with water?
Posted 10 March 2013 - 11:39 AM
Ethanol precipitates DNA in the presence of salts (usually Na+ or K+), which is why you add 2.5 volumes ethanol to a normal DNA precipitation. However, 70% is not enough to precipitate DNA, but it is enough to prevent the DNA from dissolving, while the 30% water is enough to dissolve and remove salts - hence the wash in 70% ethanol.
The usual steps for an alkaline lysis of bacteria are:
*Resuspend bacterial pellet in tris buffer with some salt and glucose (tris to maintain pH, salt for osmolarity, and glucose to keep the cells happy).
*Add lysis buffer, which is usually SDS and NaOH, both of which dissolve membranes and will denature proteins.
*Add neutralization solution which is most commonly 3M Na acetate -here the acetate neutralizes the hydroxide from the lysis step, adjusts the pH to make it favourable for binding the DNA to the silica and the Na contributes to the ionic salt concentration, which is important for DNA precipitation and precipitation of proteins. This solution may also contain chaotropic agents such as guanidine hydrochloride.
*Pellet precipitated proteins.
*Take supernate and add ethanol or isopropanol to precipitate DNA (or RNA, you can add RNase to the resuspension solution to get rid of this if you want). Here you would use a column if you are doing a column prep.
*Pellet DNA. You don't do this for a column.
*Wash in 70% ethanol
*Dry pellet to remove ethanol
*Dissolve/elute pellet in water, or 10 mM tris pH7.4 (8.4 for column elution), or TE.
Posted 10 March 2013 - 03:21 PM
RNAse is usually added at an early stage, often in the resuspension buffer. For column preps, there is no salt or glucose, just RNAse and Tris.
The neutralization is usually potassium acetate, not sodium acetate. Potassium SDS is very insoluble, and forms the white precipitate you will see after neutralization. This precipitate helps in capturing the genomic DNA released by lysis, which is otherwise indistinguishable (chemically) from the plasmid DNA you usually want. Proteins, membranes, and gDNA come out during the centrifugation of the pellet.
Water is a bad choice for resuspension, since it is usually acidic and can damage DNA. TE is ideal, both setting a basic pH, and chelating any stray magnesium to prevent accidental activation of stray DNAses.