Dan Kalderon

SMALL-SCALE PLASMID DNA PREPARATION

The fastest method is the "boiling method;" but alkaline lysis; (see large-scale preparation of plasmid DNA) using 100µl sol'n I, 200µl sol'n II and 150µl sol'n III also works well.
small scale plasmid DNA prep; plasmid prep.;
Boiling Method
Works for plasmid and M13 RF DNA

1. Grow saturated culture of plasmid-containing bacteria in 1.5ml L-broth or 2XYT containing appropriate antibiotic with good aeration at 37šC or inoculate a 1:20-1:50 dilution of TG1(or DG98) O/N with a tooth-picked M13 plaque and grow for 5 hr.

2. Transfer most of the bacterial culture to a microfuge tube and pellet bacteria(30s). Aspirate supernatant (store supernatant as a stock for M13 phage).

3. Resuspend bacteria in 100µl STET; by pipette or vortex
STET is: 8% sucrose
0.5% Triton-X100
50mM EDTA pH 8.0
10mM Tris-Cl pH 8.0

4. Add 100µl lysozyme (2mg/ml) in STET to each tube, mix and leave at room temp. for 1-5 min.

5. Place tubes in boiling water bath (switch off heat to prevent tops popping) for 1 min.

6. Spin 10 min at room temp. in microfuge.

7. Remove pellet with a flat toothpick.If the pellet occupies a large proportion of the volume of the solution continue but add STET to 200µl and, next time, use more lysozyme; if the pellet is very small and brittle, use less lysozyme.

8. Add 30µl 4M ammonium acetate, 250µl isopropanol, mix and spin 10 min at room temp in microfuge. Wash pellet with 70% ethanol, dry and dissolve in 20-50µl TE. Enough DNA is recovered for at least 5 digests but the bulk of nucleic acid is RNA which should be removed with DNase free RNase (made by boiling RNAase A at 2mg/ml for 30 mins. and cooling slowly) to see small DNA fragments. M13 R.F. preparations usually contain a number of bands of lower molecular weight than expected that presumably represent deletion products.

9. Plasmid DNA prepared in this way can be used for

(i) digestion with most enzymes
(ii) filling-in sticky ends with Klenow
(iii) purification of DNA fragments for ligation or for use as probes
(iv) nick-translation (with 32P or biotin)
(v) double-stranded sequencing
(vi) template for riboprobe synthesis

Plasmid DNA prepared as described cannot easily be succesfully used for

(i) digestion with several restriction enzymes, although digestion is normally fine if the DNA is phenol(CHCl3/IAA) extracted and ethanol precipitated (although some members of the lab find this to be untrue).
(ii) Bal 31 digestion (except with good RNase treatment as RNA inhibits Bal 31 activity on DNA)
(iii) Drosophila embryo injection
(iv) in vitro transcription

Alternative methods from step 3:

A) Add 350µl STET containing 1mg/ml lysozyme, resuspend thoroughly (1 tube at a time), boil 2 min, spin, pick out snot;(sic), add amm. acetate (this is optional) and 350µl isopropanol, spin, wash in 70& ETOH and use 1/20 in a digest.

B) As in A, but use 400µl isopropanol, skip the 70% ETOH wash, and use 1/50 per digest.