* Wear gloves when handling Neutralization Solution. Do not add bleach to sample preparation waste.
Storage and stability
ü RNase A stored at -20℃.
ü High Purity Plasmid Miniprep Kit except RNase A can be stored for up to 12 months at room temperature (15-25℃) or at 4℃ for storage periods longer than 12 months.
ü If precipitate forms in the buffers during storage, it should be redissolved by incubating the buffers at 37℃ before use.
ü After addition of RNase A, the Resuspension Solution can be used for 6 months when stored at 4℃.
Plasmid Miniprep Kit is designed for rapid and cost-effective small-scale preparation of high quality plasmid DNA from recombinant E.coli cultures. The kit utilizes an exclusive silica-based membrane technology in the form of a convenient spin column. Each spin column can recover up to 40 μg of plasmid DNA. The kit can be successfully used for efficient purification of any size plasmids and cosmids. The actual plasmid yield and optimal culture volume depend on the plasmid copy number and medium used for cultivation.
Pelleted bacterial cells are resuspended and subjected to SDS/alkaline lysis (1) to liberate the plasmid DNA. The resulting lysate is neutralized to create appropriate conditions for binding of plasmid DNA on the silica membrane in the spin column (2). Cell debris and SDS precipitate are pelleted by centrifugation, and the supernatant containing the plasmid DNA is loaded onto the spin column membrane. The adsorbed DNA is washed to remove contaminants, and is then eluted with a small volume of the Elution Buffer (10 mM Tris-HCl, pH 8.5).The purified plasmid DNA is ready for immediate use in all molecular biology procedures such as conventional digestion with restriction enzymes, PCR, transformation and automated sequencing.
1. Birnboim H.C., and Doly, J. (1979) A rapid alkaline lysis procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513 -1522.
2. Vogelstein, B. and Gillespie, D. (1979) Preparative and analytical purification of DNA from agarose. Proc. Natl. Acad. Sci. USA 76, 615-619.
Add the provided RNase A solution to the Resuspension Solution and mix. After addition of RNase A, the Resuspension Solution can be used for 6 months when stored at 4℃.
? Prior to the initial use of the kit, dilute the Wash Buffer (W) with ethanol (100%):
? After the ethanol has been added, mark the check box on the bottle to indicate the completed step.
? Check the Lysis Solution and the Neutralization Solution for salt precipitation before each use. Redissolve any precipitate by warming the solution at 37℃, then cool back down to 25℃ before use. Do not shake the Lysis Solution too vigorously.
? Both the Lysis Solution and the Neutralization Solution contain irritants. Wear gloves when handling these solutions.
? All purification steps should be carried out at room temperature.
? All centrifugations should be carried out in a table-top microcentrifuge at >12000x g (10,000-14,000 rpm, depending on the rotor type).
Growth of Bacterial Cultures
1. Pick a single colony from a freshly streaked selective plate to inoculate 1-5 ml of LB medium supplemented with the appropriate selection antibiotic. Incubate for 12-16 hours at 37°C while shaking at 200-250 rpm. Use a tube or flask with a volume of at least 4 times the culture volume.
2. Harvest the bacterial culture by centrifugation at 8000 rpm (6800x g) in a microcentrifuge for 2 min at room temperature. Decant the supernatant and remove all remaining medium.
Do not overload the column:
For high-copy-number plasmids (see Table 1), do not process more than 5 ml of bacterial culture. If more than 5 ml of such a culture are processed, the spin column capacity (40 ?g of dsDNA) will be exceeded and no increase in plasmid yield will be obtained.
For low-copy-number plasmids (see Table 1), it may be necessary to process larger volumes of bacterial culture (up to 10 ml) to recover a sufficient quantity of DNA.
Table 1 Copy numbers of various vectors
Experienced Users Procedure
1.Pellet 1–5 ml of an overnight culture (1–2 x 109 E.coli in LB medium). Thoroughly remove all medium from the cell pellet.
2.Resuspend the pelleted cells in 250 μl of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
Note. Ensure RNase A has been added to the Resuspension Solution.
3.Add 250 μl of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 minutes to avoid denaturation of supercoiled plasmid DNA.
4.Add 350 μl of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris.The neutralized bacterial lysate is cloudy and viscous.
5.Centrifuge for 10 min at 14,000× g (12,000rpm) to pellet cell debris and chromosomal DNA.
6.Transfer the supernatant to the supplied spin column by decanting. Avoid disturbing or transferring the white precipitate.
7.Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
Note. Do not add bleach to the flow-through.
10.Repeat the wash procedure (step 7) using 500 μl of the Wash Solution (W).
11.Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
12.Transfer the spin column into a fresh 1.5 ml microcentrifuge tube (not included). Add 50 μl of the Elution Buffer to the center of spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min at 14,000× g (12,000rpm).
Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%.For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70℃ before applying to silica membrane.
13.Discard the column and store the purified plasmid DNA at -20℃.