Plasmid Isolation with BioBasic Miniprep Kit

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1. On the day before your minipreps you will use sterile technique near a Bunsen burner to set up 3ml of LB culture with 50ug/ml of kanamycin (or other appropriate antibiotic) (add 5ul of the 50mg/ml stock solution) for each colony of interest. Using a sterile P200 pipette tip touch one colony from your patch plate and then drop it into one culture tube. Be careful to record which culture corresponds to which patch on your patch plate. Place cultures (with lids at the loose setting) to grow overnight in a 37oC shaking incubator.

2. From your overnight E. coli culture, spin down 1.5mL in a centrifuge tube at 12,000xg for 2 min and decant off supernatant.

3. [This protocol is adapted from the BioBasic EZ-10 Spin Column miniprep kit.] Add 100 µl of Solution I to the cell pellet. Mix well and incubate for 1 min at room temp.

4. Add 1 µl of VisualLyse solution to the mixture above. Note: VisualLyse is not necessary for the reaction but serves as a visual indicator of lysis to prevent under or over incubation in the following step.

5. Add 200 µl of Solution II to the tube and mix gently by inverting the tube 4-6 times (do not vortex). The VisualLyse in the solution will turn blue in this step. Your sample should be homogenous in color–if you see uneven color or white/brown cell clumps, continue to invert the tube to gently mix. Incubate at room temperature for 1 min.

6. Add 350 µl of Solution III and mix gently by inverting the tube until all traces of blue color have become colorless. Incubate at room temperature for 1 minute. You should see a fluffy white precipitate form, and the lysate should become less viscous.

7. Centrifuge at 12,000rpm for 5 min.

8. Transfer the above supernatant to the EZ-10 Column in a collection tube.

9. Centrifuge at 10,000rpm for 2 minutes.

10. Discard the flowthrough in the tube. Add 750 µl of Wash Solution to the column and centrifuge again at 10,000rpm for 2 min.

11. Repeat step 10 for a second wash. Discard the flowthrough in the collection tube.

12. Without adding anything else to the column, centrifuge at 10,000rpm for 2 minutes to remove any residual wash solution that remains in the column.

13. Discard the collection tube and transfer the column to a clean 1.5ml centrifuge tube.

14. To elute DNA from the column, add 50 µl of PCR grade water into the center of the column and incubate at room temperature for 2 min.

15. Centrifuge at 10,000rpm for 2 min.

Following this protocol, you should have extracted plasmid in 50 µl of water. Before transforming M. xanthus with this plasmid, you need to ensure the plasmid is at the proper concentration (proceed to nanodrop step and then store your plasmid at -20C.