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°C water bath until the solution clears.
The Plasmid MaxiPrep Kit provides a rapid method for preparation of large quantities of high quality, endotoxin-free plasmid DNA. Purification is based on silica spin filter technology. The silica membrane binds DNA under high salt concentrations and releases the bound DNA under low salt and slightly alkali conditions. The purified DNA is free of endotoxin, genomic DNA, RNA and cellular proteins and can be use directly in transfection applications. The Plasmid MaxiPrep Kit is able to purify up to 400-1,000μg of plasmid DNA from 100 mL of bacterial culture containing a high copy number plasmid .
Endotoxin-free Plasmid：Less than or equal to 0.1 EU/μg of purified plasmid DNA.
Fast and easy processing：purification takes only 40 min.
High yield：Up to 400-1,000μg of plasmid DNA can be purified from 100 ml of bacterial culture.
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 minutes.
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, until a clear lysate appear.
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 minutes at room temperature. Centrifuge at ~12,000x g for 2containing RNase A to the pellet and vortex until homogeneous. Incubate the lysate at room temperature for 5 to 10 minutes.
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 once with 10 ml Wash Buffer PX. Centrifuge at ~12,000x g for 2 min. Discard the flow-through.
10.Wash the column with 10 ml Wash Buffer W, Centrifuge at ~12,000x g for 2 min. Discard the flow-through.
11.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.
12.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 minutes at room temperature. Centrifuge at ~12,000x g for 2 min. 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.