PureYield Plasmid miniprep
Updated 10/13/2020 12:18 PM
Introduction
Materials
- PureYield Plasmid miniprep Kit
Procedure
Procedure
- Transfer 600µL of bacterial culture grown in LB medium to a 1.5mL microcentrifuge tube. Note: If you wish to process larger volumes of bacterial culture (up to 3.0mL), use the protocol provided in Section 4.C.
- Add 100µL of Cell Lysis Buffer, and mix by inverting the tube 6 times. The solution should change from opaque to clear blue, indicating complete lysis. Note: Proceed to Step 3 within 2 minutes. Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, process samples in groups of ten or less. Continue with the next set of ten samples after the first set has been neutralized and mixed thoroughly.
- Add 350µL of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting the tube. The sample will turn yellow when neutralization is complete, and a yellow precipitate will form. Invert the sample an additional 3 times to ensure complete neutralization.
- Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
- Transfer the supernatant (~900µL) to a PureYield™ Minicolumn. Do not disturb the cell debris pellet. For maximum yield, transfer the supernatant with a pipette.
- Place the minicolumn into a PureYield™ Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds.
- Discard the f owthrough, and place the minicolumn into the same PureYield™ Collection Tube.
- Add 200µL of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds. It is not necessary to empty the PureYield™ Collection Tube.
- Add 400µL of Column Wash Solution to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.
- Transfer the minicolumn to a clean 1.5mL microcentrifuge tube, then add 30µL of Elution Buffer directly to the minicolumn matrix. Let stand for 1 minute at room temperature. Notes: 1. Nuclease-free water at neutral pH can also be used to elute DNA. 2. For large plasmids (>10kb), warm the Elution Buffer to 50ºC prior to elution, and increase elution volume to 50µL. Also incubate the column at room temperature (22–25°C) for 5–10 minutes before proceeding to Step 11.
- Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge tube, and store eluted plasmid DNA at –20°C.
"The system provides a rapid method to purify plasmid DNA using a silica membrane column. Plasmid DNA can be purified in less than 10 minutes, depending on the number of samples processed. The PureYield™ Plasmid Miniprep System incorporates a unique Endotoxin Removal Wash to remove protein, RNA and endotoxin contaminants from purified plasmid DNA." ~ from Promega