Team:LMU-Munich/Notebook/Protocols/4 Plasmid extraction from cells

(Source: http://www.promega.com/tbs/tb374/tb374.pdf)

4.A. Centrifugation Protocol

The following procedure is performed at room temperature.

1. 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.

2. 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.

3. 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.

4. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.

5. Transfer the supernatant (~900μl) to a PureYield™ Minicolumn.

Do not disturb the cell debris pellet. For maximum yield, transfer the supernatant with a pipette.

6. Place the minicolumn into a PureYield™ Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds.

7. Discard the flowthrough, and place the minicolumn into the same PureYield™ Collection Tube.

8. 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.

9. Add 400μl of Column Wash Solution to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.

Add new 1.5 ml microcentrifuge tube and microcentrifuge dry again for 1 minute

10. 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.

11. Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute the plasmid DNA.

Eluate a second time with 20 µl Elution Buffer

Cap the microcentrifuge tube, and store eluted plasmid DNA at –20°C.

4.C. Alternative Protocol for Larger Culture Volumes

This alternative protocol can be used with the PureYield™ Plasmid Miniprep System to harvest and process up to 3ml of bacterial culture. We recommend using 3.0ml of bacterial culture when using low-copy-number plasmids.

1. Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed in a microcentrifuge.

2. Discard the supernatant.

3. To process a total of 3.0ml of culture, add an additional 1.5 ml of bacterial culture to the same tube. Repeat Steps 1 and 2.

4. Add 600μl of TE buffer or water to the cell pellet, and resuspend completely.

5. Proceed to Step 2 of the PureYield™ Plasmid Miniprep System protocol (Section 4.A).