On-line Methods Ten-minute miniprep

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19 April 2002 updated 22 March 2018

 

Mike's hints for successful minipreping.

  1. Always, always, always mark each culture tube in an unambiguous manner (with a sharpie pen).
  2. Always, always, always mark each collection and final collection tube in an unambiguous manner (with a sharpie pen). These should correspond to your cultures - all tubes look identical.
  3. If you fail to recover plasmid, the most likely cause is that you failed to add ampicillin or that your ampicillin was bad. Repeat using a fresh stock of ampicillin.
  4. Always check minipreps by running out 2-4 µL on an agarose gel before setting up restriction digests, etc

Protocol  Zhou et al 1990 as modified by Tamara Basta

  1. Spin 1.5 ml of an overnight bacterial culture for 1 min. (double if the culture is growing slowly)
  2. Gently decant supernatant - leave ~50µL of supernatant with the cell pellet
  3. Resuspend by running tube along tube rack (Alberto's Trick)
  4. Add 300 µL of TENS (lysis buffer) and mix by inversion* until completely lysed -- make up TENS buffer fresh from 10 x stocks (see below).
  5. Add 150 µL 3.0 M sodium acetate, pH 5.2 and invert to mix*
  6. Centrifuge for 5 minutes pellet cellular debris and chromosomal DNA
  7. Transfer supernatant to fresh microfuge tube
  8. Add 0.9 mL -20°C 100% ethanol
  9. Spin for 5 minutes at 4°C to pellet plasmid DNA
  10. Discard supernatant and rinse pellet twice with 70% ethanol
  11. Dry pellet in speed-vac for ~5 minutes
  12. Resuspend pellet in 50-100 µL in resuspension buffer (you can add RNAase at this step).

* do not vortex at these steps, it will shear the chromosomal DNA

TENS Buffer:  TE + 0.1N NaOH and 0.5% SDS from 10X TE, 5N NaOH and 20% SDS


Qiagen miniprep

  1. Grow up single clone in 5ml LB + appropriate antibiotic
  2. Centrifuge 1.5 ml of an overnight bacterial culture for 1 min. (double if the culture is growing slowly)
  3. Gently decant supernatant - resuspend cell pellet in 250 μL P1 buffer + RNase
  4. Resuspend by running tube along tube rack (Alberto's Trick)
  5. Add 250 μL Buffer P2 and mix thoroughly (but gently) by inverting the tube 4–6 times until the solution becomes viscous + clear. Do not vortex.
  6. Neutralize by adding 350 μl Buffer N3 and mix immediately by inverting the tube several times.
  7. In microfuge, spin for 10 minutes to pellet cellular debris and chromosomal DNA
  8. Transfer the supernatant the blue QIAprep spin column, avoid disturbing the pellet - spin 1 minute
  9. Discard flow through - wash column with 0.75 ml Buffer PE – centrifuge for 1 min.
  10. Discard the flow through, spin again for 1 minute
  11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube
  12. Elute DNA into the clean tube by adding 50 μL EB Buffer (10 mM Tris·Cl, pH 8.5) to the center of QIAprep spin column, let stand for 1 min, and centrifuge for 1 min -
  13. To check plasmid - run out 1-2 μL on agarose gel.
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