Miniprep Plasmid DNA for Bio113

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Day Before Lab Day (4:30 pm) For each miniprep, grow 2 mL of LB + ampicillin culture, 37° C, overnight (O/N); shake at 250 RPM to make sure the cultures are well aerated.

Lab Day

  1. Label two 1.5 mL tubes with your initials and the clone you will miniprep (e.g., X1).
  2. Add 600 µL of O/N culture to an appropriately labeled 1.5 mL microfuge tube. Save the rest of the O/N culture and keep them sterile.
  3. Add 100 µL 7X Lysis Buffer (blue color). Mix by inverting the tube 4-10 times. Solution should become clear blue instead of opaque. Proceed to the next step within 3 minutes.
  4. Add 350 µL of Neutralization Buffer (yellow color; RNase A already added) and mix by inverting the tube until the entire solution and precipitate is yellow. This buffer is stored at +4 ° C.
  5. Microfuge full speed for 2 minutes at room temperature (RT°).
  6. While your samples are spinning, prepare Zymo-Spin II column (with white binding resin) by inserting into 2 mL collection tube. Be sure to label the spin column and the collection tube. Also label a second 1.5 mL microfuge tube for the final elution.
  7. Transfer ~900 µL yellow supernatant to Zymo-Spin II column. Do not transfer any of the solid precipitate. Better to leave some clear yellow liquid behind than to get greedy and transfer some solid material.
  8. Microfuge full speed for 2 minutes at RT°.
  9. Discard liquid flowthrough and reinsert Zymo-Spin II column into same collection tube.
  10. Add 200 µL Endo-Wash Buffer to the Zymo-Spin II column. Spin full speed for 15 seconds at RT°. Repeat this step a second time. No need to empty flow through until second wash is completed.
  11. Add 400 µL Zyppy Wash Buffer (with ethonol already added). Spin full speed for 30 seconds at RT°. Discard liquid flowthrough. Repeat this step a second time. Discard liquid flowthrough and and the 2 mL collection tube after the second wash. The DNA is still in the spin column.
  12. Transfer Zymo-Spin II column to a clean and appropriately labeled 1.5 mL microfuge tube (from step 5 above). Cut the lid off this tube before microfuging!
  13. Add 30 µL Zyppy Elution Buffer to the center of the to the Zymo-Spin II column. Let it stand for 1 minute to maximize yield.
  14. Microfuge full speed for 30 seconds at RT°. SAVE THE LIQUID with your plasmid. Discard the spin column.
  15. You can NanoDrop the DNA to determine the concentration.