Before Starting for MaxiPrep Procedures
ü Add RNase A to the Solution I according to instructions on the label. Mix well. Mark on the label that RNase A is added. Store at 4℃.
ü If the Solution II contains salt precipitates, warm the buffer in a 37℃ water bath until the solution clears.
ü Add 1.5 volume of 96 - 100% ethanol to 1 volume of Wash Buffer W, Mix well. Mark on the labels that ethanol is added. Store both wash buffers with ethanol at room temperature.
Plasmid Maxiprep Kit is designed for rapid and cost-effective large-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 400-1,000 μg of plasmid DNA from 100 ml overnight bacterial culture. 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.
The method is based on silica-membrane spin column, silica particles bound DNA selectively athigh salt concentration and low pH, while protein and other contaminants are wash to remove. The pure DNA is eluted with TE buffer or water. This method requires few manipulations, and is both faster and easier to perform than other organic-based extraction methods. The purified DNA is suitable for all common molecular biology procedures, including restriction digestion, cloning, sequencing, etc.
High yield : 100 ml suspension yield 400-1,000 μg plasmid DNA of high copy.
High purity : A260/A280=1.7-1.9. no chloroform and phenol, which is safe for people.
Fast : purification takes only 40 min.
RNase A: store at -20℃. Before starting, please add RNase A to Solution I, mix well and store at 4℃.
Other reagent can be store at room temperature.
If precipitate forms in the buffers during storage, it should be redissolved by incubating the buffers at 37℃ before use.
1.Grow transformed E. coli in LB medium.
2.Pellet 100 ml (high copy number plasmid) or 250–500 ml (low copy number plasmid) of an overnight culture.
3.Add 7.5 ml Solution I containing RNase A to the pellet and vortex until homogeneous. Incubate the lysate at room temperature for 5 to 10 min.
4.Add 7.5 ml Solution II. Mix gently by inverting the capped tube twelve times. Do not vortex. Incubate the lysate at room temperature for 3 to 5 min.
5.Add 10 ml Solution III. Mix immediately by inverting the capped tube until the mixture is homogeneous. Do not vortex.
6.Centrifuge at ~12,000x g for 12 min at room temperature.
7.Carefully remove and load the supernatant from Step 6 onto the spin column. Incubate for 5 min at room temperature. Centrifuge at~12,000x g for 2 min. Discard the flow-through.
8.Wash the column once with 10 ml Wash Buffer PB. Centrifuge at ~12,000x g for 2 min. Discard the flow-through.
9.Wash the column with 10 ml Wash Buffer W, Centrifuge at ~12,000x g for 2 min. Discard the flow-through. Repeat Step 9 again.
10.Centrifuge the column at ~12,000x g for 4 min to remove any residual Wash Buffer W. Discard the Wash Tube with the flow-through. Air dry the column for 5 minutes.
11.Place the Spin Column in a clean 50-ml Recovery Tube. Add 1.5 to 2 ml of preheated Eluent Buffer to the center of the column. Incubate the column for 5 minute at room temperature. Centrifuge at ~12,000x g for 2 minutes. The Recovery Tube contains your purified plasmid DNA. Discard the column. Store the plasmid DNA at -20℃.
Perform DNA quantitation using UV absorbance at 260 nm.
1. Prepare a dilution of the DNA solution. Mix well. Measure the absorbance at 260 nm (A260) of the dilution in a spectrophotometer (using a cuvette with an optical path length of 1 cm) blanked against the dilution buffer.
2. Calculate the concentration of DNA using the formula:
DNA (μg/ml) = A260× 50 × dilution factor
For DNA, A260 = 1 for a 50 μg/ml solution measured in a cuvette with an optical path length of 1 cm.