My understanding of the plasmid isolation process is that we use the alkaline lysis buffers P1,P2 and P3 and centrifuge the clarified floccules which are a mixture of K-SDS, Chromosomal DNA and Plasmid DNA in addition to proteins. I recently used the Qiagen GIGA kit ,which comes with the filtration apparatus(Nalgene; Nylon 5 um pore size). On the same line of thought, I was wondering if we could use a nylon filter and pass the clarified lysate and subsequently use a silica based column for plasmid DNA extraction. ANy thought/two cents on this?
Thanks!
Arjun
Dear Arjun,
Alkaline lysis method using P1 P2 & P3 is a miniprep protocol for purifying smaller amounts of cloned plasmid from smaller volume of cultures. The chemistry used in this protocol is standardized to work with smaller amounts of bacterial culture (typically 3-5 mL) and yields 10-50 ug of relatively pure plasmid DNA, which is sufficient for most of the regular downstream application in a molecular biology. However, Giga scale preparation is followed for purification of larger volumes of bacterial cultures (typically 2-5 Litres) mostly for industrial applications. Hence additional filtration is required to separate large amounts of chromosomal DNA & protein complexes from the plasmid DNA and there comes the 5 um filter.
Therefore I think that additional filtration with a 5um filter in miniprep is not going to make significant difference in your preparation. You can directly proceed with the P3 supernatant for Isopropanol mediated plasmid DNA purification or to silica-column based purification.
best wishes,
SD
Dear Arjun
- Yes, you can filter away the gelatinous material
- Indeed it is a good idea to filter this away as any gelatinous material in your supernatant could block the silica column and prevent elution of your RNA
- In the old days before columns we would filter away such material with Muslin before subjecting to cold ethanol precipitation
In case you haven't already tried this or had problems, the filtration method works fine as long as 1) the mixing after the P3 addition is such that the preparation has a flocculant texture (I tell people like Chinese Egg Drop Soup) rather than being viscous with genomic DNA , 2) after adding the prep to the syringe or vacuum filter you let it sit for 10 or 15 minutes for the precipitates to float to the top so the membrane doesn't immediately come into contact with solids and clog, and 3) you don't apply too much pressure such that the genomic DNA bound up in the KDS precipitate doesn't contaminate the plasmid containing clarified liquid.
Dear Sib,
While trying to reverse engineer the Giga Scales may not be feasible, I was thinking of Maxi. Please note, I am not talking about the DEAE-Silica column but the Silica column used for a much larger scale(Eg.Gencatch Econospin). and yes, I am not talking about mini-preps.
@Laurence,
Thank you for your comments.
Dear Christopher,
Precisely! I always look for a "dessicated coconut" appearance.
Based on the three comments, I'll wager that , theroteicaly, this method should give us yields of the maxi levels.
Thanks!
Arjun
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