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These are just the instructions for the Qiagen miniprep kit we use, which can be downloaded online (search for 'qiagen miniprep protocol'), but if you don't want to download a giant PDF and sift through it looking through the one line you need, it's also here!

 

In general: 5 mL LB incubated overnight, or 2-3 mL LB30 incubated for 8+ hours (overnight is also fine), should be sufficient for most purposes. If you are doing multiple sequencing reads on a medium or low-copy plasmid, or something else that requires a lot of DNA, you may want to double the volume and do two simultaneous minipreps.

Centrifuge miniprep protocol:

  1. Pellet cells from culture in centrifuge (5 minutes at 3500 rcf is fine).
  2. Pour out/aspirate the supernatant and resuspend the pelleted cells in 250 ul P1 buffer (on door of fridge).
  3. Add 250 ul P2 buffer and mix by inverting the tube 4-6 times. (Do not let this stage of the reaction go longer than ~5 minutes.)
  4. Add 350 ul N3 buffer and mix by inverting 4-6 times.
  5. Centrifuge for 10 minutes at ~17900 rcf. A white pellet should form at the bottom.
  6. Extract the supernatant with a pipette and apply it to a spin column (the blue EconoSpin columns; the Zymo columns should do in a pinch but are not ideal.) Avoid taking up any of the pellet or mixing it with the supernatant. Avoid adding more than ~800 ul to the column at once–either discard the excess or run the next spin cycle multiple times, discarding the flow-through after each cycle.
  7. Centrifuge for 30-60 s. Discard the flow-through.
  8. (optional) Add 500 ul PB buffer to the spin column and centrifuge for 30-60 s. Discard the flow through. (This is to get rid of trace nuclease activity, particularly in endA+ strains. It is not necessary for our standard cloning strains.)
  9. Add 750 ul PE buffer to the spin column and centrifuge for 30-60s. Discard the flow-through.
  10. Centrifuge again for 30-60 s to remove excess liquid from the column.
  11. Place the top part of the column in a clean microcentrifuge tube and add 50 ul EB buffer or water to the center of column. (You could use as little as 15-20 ul, less with the Zymo columns, although this may decrease total DNA yield somewhat.) Do not touch the surface of the column with your pipette tip, but make sure that the water is deposited directly on the column.
  12. Let stand for 1 minute.
  13. Centrifuge 30-60 s.

I generally get 300-800 ng/ul for high-copy plasmids, 30-100 ng/ul for medium-copy plasmids.

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