PLASMID MINIPREP

Before starting:

• Add RNase A to Resuspension Buffer (R3) according to the instructions on the label; mix well
• Mark on the label that RNase A is added. Store Buffer R3 with RNase A at 4 DEGC
• Warm Lysis Buffer (L7) briefly at 37 DEGC to redissolve any particulate matter
• Add 100% ethanol to Wash Buffer (W9) and Wash Buffer (W10) according to the instructions on each label; mix well
• Store wash buffers with ethanol at room temperature

1. Harvest
• Sediment the cells by centrifuging 1 - 5 mL of the overnight LB culture (use 1-2 x 10⁹ E.coli cells for each sample)
• Remove all medium
2. Resuspend
• Add 250 μL Resuspension Buffer (R3) with RNase A to the cell pellet
• Resuspend pellet until it is homogeneous
3. Lyse
Note: This step is time sensitive.
• Add 250 μL Lysis Buffer (L7)
• Mix gently by inverting the capped tube five times (Do NOT vortex)
• Incubate the tube at room temperature for 5 minutes
4. Precipitate
• Add 350 μL Precipitation Buffer (N4)
• Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous (Do NOT vortex)
• Centrifuge the lysate at >12,000 X g for 10 minutes
5. Bind
• Load the supernatant from step 4 onto a spin column in a 2-mL wash tube
• Centrifuge the column at 12,000 X g for 1 minute
• Discard the flow-through and place the column back in the wash tube
6. Wash and Ethanol Removal
• Add 700 μL Wash Buffer (W9) with ethanol to the column
• Centrifuge the column at 12,000 X g for 1 minute
• Discard the flow-through and place the column back into the wash tube
• Centrifuge the column at 12,000 X g for 1 minute
• Discard the was htube with the flow-through
7. Elute
• Place the spin column in a clean 1.5-mL recovery tube
• Add 75 μL of preheated TE Buffer (TE) to the center of the column
• Incubate the column for 1 minute at room temperature
8. Recover
• Centrifuge the column at 12,000 X g for 2 minutes
• The recovery tube now contains the purified plasmid DNA
• Discard the column
• Store plasmid DNA at 4 DEGC (short-term) or store the DNA in aliquots at -20 DEGC (long-term)

Thank you to Life Technologies for this protocol.

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