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 Add 1.5 volume of 96%-100% ethanol to 1 volume of Wash Buffer W. Mix well. Mark on the label that ethanol is added.
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 method is based on silica adsorption, silica particles bound plasmid DNA selectively at high 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.
Flexibility : easily scale up or down.
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 for 5 to 10 minutes at room temperature.
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 minutes.
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 a new 50-ml centrifuge tube. Add Silica Powder Suspension at a ratio of 1.2 ml of Suspension per 25 ml supernatant. Mix thoroughly by inverting 6 times. Incubate for 5 minutes at room temperature.
8.Centrifuge at ~12,000x g for 2 min. Discard the flow-through.
9.Wash the column once with 10 ml Wash Buffer PB, resuspend the precipitates by vortexing. Centrifuge at ~12,000x g for 2 min. Discard the flow-through.
10.Wash the column with 10 ml Wash Buffer W, resuspend the precipitates by vortexing.Centrifuge at ~12,000x g for 2 min. Discard the flow-through. Repeat Step 9 again.
11.Pipetting away any residual Wash Buffer W, open the cap, air dry the tube for 30 minutes or place the tube in a super clean bench to dry for 15 minutes.
12.Add 1.5 to 2 ml of preheated Eluent Buffer to the center of the tube. Resuspend the precipitates by vortexing Incubate the column for 5 minute at room temperature. Centrifuge at ~12,000x g for 2 min. The supernatant contains your purified plasmid DNA. Transfer the supernatant to a new tube. 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.