Quick Plasmid Miniprep

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Quick Plasmid Miniprep

Quick Plasmid Miniprep
by Michael Koelle, modified from Holmes & Quigley (1981) Anal. Biochem.114, 193-197.

Link: http://www.med.yale.edu/mbb/koelle/

1. Grow 2 ml bacteria 5hrs to o/n at 37°. TG1 needs only 5-6 ours to give a dense 2 ml culture starting from a colony. DH5 requires at least 10 hours.

2. Pour into 1.5 ml eppie. (Can save remaining culture for possible large scale growth later.)

3. Pellet bacteria 30 sec in microfuge. Aspirate sup using a P200 tip jammed on to the end of the aspirator tube.

4. Suspend in 200 l STET by vortexing. Add 20 l fresh 10mg/ml lysozyme (can add these both together)

5. Place in boiling water bath 40 sec. (Kills DNAses and ppts protein and chromosomal DNA).

6. Centrifuge immediately 5-10 min in fuge (4° or 25°).

7. Remove large clot with toothpick. Precipitate with 200 l 2-propanol: room temp 5-10 min, and spin 5 min in fuge. Get a fairly large visible ppt. Aspirate sup., careful not to lose pellet.

8. Wash pellet with 70% EtOH, aspirate sup. Dry briefly in speed vac (don't dry completely). Resuspend in 50 l TE.

9. Restriction digest: use 2 l DNA in a 20 l reaction. After digest, add RNase A to about 0.01 g/l 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 pipetter (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 explaination, 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 l of the water, and proceed with the prep as usual (except add ~500 l isopropanol instead of 200 l) 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 l 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.

------------------------------------------------------------------------
Medium
Luria broth + 50-100 g/ml carbenicillin

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

Lysozyme (Sigma) - 10mg/ml

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