boiling miniprep - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

Quick Plasmid Miniprep by boiling

This protocol was copied from one source online using this link which is now defunct. The original online protocol was written by Michael Koelle, modified from Holmes & Quigley (1981) Anal. Biochem.114, 193-197.

  1. Grow a 1-2 mL culture of bacteria 5 hrs to o/n at 37°.

  2. Transfer 1 mL of culture into a 1.5 ml Eppendorf tube. (Save remaining culture as it can be used for larger scale culture later.)

  3. Pellet bacteria 20-30 sec in microcentrifuge. Remove supernatant by decantation.

  4. Suspend cells in 200 µl STET buffer (see below for recipe) by vortexing. Add 20 µl fresh 10 mg/ml [Lysozyme] (STET and lysozyme can be added together).

  5. Place in boiling water bath for 40 seconds. This treatment inactivates DNAses, and denatures proteins and chromosomal DNA.

  6. Centrifuge immediately 5-10 min in microcentrifuge (4°C if available else centrifuge at room temperature).

  7. Remove the large clot at the bottom of the tube with a fresh toothpick.

  8. Add 200 µl 2-propanol and incubate for 5-10 min at room temperature.

  9. Spin 5-10 min in a microcentrifuge. You should get a fairly large visible precipitation.

  10. Remove supernatant with a P1000 pipette, be careful not to lose pellet.

  11. Wash pellet with 500 µL 70% EtOH and remove supernatant by decantation.

  12. Spin tube again briefly to collect ethanol in the bottom of the tube. Remove all ethanol with a P20 or P200 pipette.

  13. Dry DNA by letting the tube stand with an open lid on the bench for 15 min.

  14. Resuspend DNA pellet in in 50 µl 1x TE buffer.

short version.

To analyze by restriction digest: use 2 µl DNA in a 20 µl reaction. After digest, add RNase A to about 0.01 µg/µl for a few seconds before loading on gel. This is best accomplished by making some DNA gel loading buffer containing 0.1 mg/ml RNase A and mixing this with the digest just before loading. The tube of RNAse-containing loading buffer can be left out on the bench and reused for months before the RNase goes bad.

Notes:

Boiling preps are faster than alkaline lysis minipreps mainly because you never have to transfer the samples to new tubes (thus you only have to label one set of tubes). The DNA from boiling preps is somewhat dirtier, but is perfectly adequate for restriction digests.

If you're doing lots of minipreps at once, an Eppendorf repeater pipette (uses "combitips") saves a lot of time and aggravation. Another great device is the "multi tube vortexer" from VWR, for easily resuspending lots of bacterial pellets simultaneously.

A few E. coli strains (e.g. "BB4" cells from Stratagene) simply don't work for boiling minipreps. Maniatis says these strains have a heat resistant nuclease, but I doubt that is the correct explanation, since the EDTA present in all the buffers should kill any DNAse activity. In any case, these strains offer no advantages; don't use them.

The most convenient boiling water bath: buy a small electric deep fat fryer from a kitchen gadgets store (~$25). Plug it in and it's boiling in ~4 min.

Some brands of "eppendorf" style 1.5 ml snap cap tubes have loose fitting caps that pop open in the boiling water bath. If you use a good brand this doesn't ever happen. Even if the cap pops and the tube falls into the bath and fills with water, you can just pick the tube out, remove all but ~500 µl of the water, and proceed with the prep as usual (except add ~500 µl isopropanol instead of 200 µl) and it will work just fine. Some brands of tubes don't pop because they have tiny holes in the middle (e.g. Applied Scientific). This is great in the boiling prep, but the samples will evaporate in the fridge over a period of ~1 month, so these tubes aren't great.

An oddity of this prep is that it will not work as a template for PCR reactions perhaps it contains some type of inhibitor of Taq polymerase. If you need to PCR using your plasmid as a template, a convenient source of material to use 1 µl of your bacterial culture. No special steps need to be taken to lyse the bacteria. Using about 25-30 rounds of amplification you will get great yields.

STET buffer (50 mL)

  • 8% sucrose (6.6 ml 60%)
  • 5% Triton X-100 (2.5 mL 100%)
  • 50 mM EDTA (5.0 mL 0.5 M)
  • 50 mM Tris pH 8.0 (2.5mL 1 M)
  • ddH2O 33.3 ml
  • 50.0 ml total

Lysozyme (Sigma) - 10 mg/ml

RNase A - 10 mg/ml in TE, heat in a boiling waterbath a few minutes (to kill contaminating DNAses), and store frozen.

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