The Clontech NeucleoSpin Plus Plasmid Miniprep Kit is used in our laboratory to purify high quality plasmid DNA for a variety of uses including cloning and sequencing. This kit is a quick and efficient way to purify plasmid DNA from small culture volumes (about 2 ml) of bacterial cells - in our case we are mainly purifying plasmid DNA from DH5alpha or MC1061 cells. This kit utilizes disposable spin columns that contain an activated silica membrane to isolate and separate the nucleic acids from proteins and other cellular components. These columns can also be used with vacuum filtration if dealing with many preps.
The Clontech NeucleoSpin Plus Plasmid Miniprep Kit includes: 50 Spin columns, 50 2-ml collection tubes, re-suspension buffer (Buffer A1), lysis buffer (Buffer A2), binding buffer (Buffer A3), wash buffer (Buffer A4), optional wash buffer (Buffer AW), elution buffer (Buffer AE),and RNase A. The RNase comes in a lyophilized form, so it must resuspended in Buffer A1. The only components not included but required are ethanol and 1.5 ml microcentrifuge tubes.
The protocol is straightforward: the bacterial cell pellet from about 2 ml of an overnight culture is mixed in resuspension buffer (Buffer A1) and lysed with alkaline/SDS containing lysis buffer (Buffer A2), releasing the plasmid DNA into solution, while the genomic DNA and other cellular debris are precipitated out of solution. The binding buffer (Buffer A3) is added, and the cell/buffer mixture is placed on ice. Then, the supertanant (cleared via a quick spin to remove the precipitated debris) is placed into the spin column inserted into the collection tubes. The special silica membranes in the filter are designed to trap the plasmid DNA while other contaminants are allowed to flow through. The filter membrane is first washed with Buffer AW (washing with Buffer AW is not required, but we recommend it). This extra washing step is recommended if the bacterial strain has high nuclease activity or if the plasmid DNA will be sequenced. The filter membrane in the spin column is further washed with wash buffer (Buffer A4), which must be mixed with ethanol. Then the DNA of interest is eluted off the filter membrane with a low-salt Tris elution buffer (Buffer AE), or TE can be used. We recommend from our experience that the plasmid DNA is eluted twice from the filter membrane to increase yield. From a 2-ml bacterial culture, one can obtain 2-3 ug/ml plasmid DNA depending on the amount of cells from overnight culture. Based on A260/A280 readings (as well as analyses of the DNA on an agarose gel) the purified DNA is very clean with very little contaminants, if any.
The only minor problems that we have encountered are that: 1. the SDS in the lysis buffer (Buffer A2) tends to precipitate when stored for prolonged time at room temperature, so it has to be warmed up in a water bath prior to use to re-dissolve the SDS in the solution and 2. the binding buffer (Buffer A3) contains salt which can precipitate if left on ice too long. Also, in some bacterial strains, it is hard to lyse the cells, so the pellet should be resuspended and incubated with resuspension buffer containing lysozyme, which is not included in the kit.
Overall, we are pleased with this product and have no problems recommending it. We have used it to purify plasmid DNA quickly and have been happy with the quality of the DNA that we have obtained.
Hee Chul Lee
Graduate Student
Department of Biochemistry
NYU School of Medicine