Team:TU Dresden/Notebook/Protocols/HiYieldPrestoMiniPlasmidKit

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HiYield Presto Mini Plasmid Kit

1. Harvesting: Transfer 1.5 mL of cultured bacterial cells to a 1.5 mL microcentrifuge tube. Centrifuge at 14-16,000 g for 1 minutes at room temperature to form a cell pellet. Discard the supernatant completely; use narrow pipette tip to ensure removal.
2. Resuspension: Transfer 1.5 mL of cultured bacterial cells to a 1.5 mL microcentrifuge tube. Centrifuge at 14-16,000 g for 1 minutes at room temperature to form a cell pellet. Discard the supernatant completely; use narrow pipette tip to ensure removal.
3. Cell lysis: Add 200 μL of PD1 Buffer (make sure RNAse A is added) to a new 1.5 mL microcentrifuge tube. Add 2 μL of TrueBlue Lysis Buffer to the same tube and mix by shaking gently. Transfer the mixture to the 1.5 mL microcentrifuge tube containing the cell pellet. Resuspend cell pellet completely by vortex or pipette.
4. Neutralization: Add 300 μL of PD3 Buffer then mix immediately by inverting the tube 10 times. Do not vortex to avoid shearing. Then centrifuge the 1.5 microcentrifuge tube at 14-16,000 g for 3 minutes at room temperature.

5. Wash: For Improved Downstream Sequencing Reactions: Add 400 μL of W1 Buffer into the PD Column. Centrifuge at 14-16,000 g for 30 seconds. Discard flow-through from the Collection Tube and place the PD Column back afterwards.

   For Standard Plasmid DNA Purification: Add 600 μL of Wash Buffer (make sure absolute ethanol was added) into the PD Column. Centrifuge at 14-16,000 g for 30 seconds at room temperature. Discard the flow through then the place the PD Column back in the 2 mL Collection Tube. Centrifuge at 14-16,000 g for 3 minutes at room temperature to dry the column matrix. Transfer the dried PD Column to a new 1.5 mL microcentrifuge tube.

6. Elution: Add 50 μL of Elution Buffer, TE or water into the CENTER of the column matrix. Let stand for at least 2 minutes to allow it to be completely absorbed. Centrifuge at 14-16,000 g for 2 minutes at room temperature to elute the purified DNA.