Improved-Home-Made-Plasmid-Mini-Prep

SI: 50 mM Tris-HCl, 10 mM EDTA, pH 8.0 (TE buffer), with 100 ug/ml RNase and lyseblue

SII: 0.2M NaOH and 1% SDS

SIII: 2.0M HAc, 0.6M KAc, 0.4M MnCl2, 0.4 M CaCl2.

N3:

3.0 M KOAc, pH 5.5 (29.5 g/ 100ml water, adjust pH with glacial acetic acid )

or: 4.2 M guanidine hydrochloride, 0.9M KOAc, pH 4.8, adjust to a final concentration of 1.75 M GuHCl

or Qiagen N3: 3M KOAc, 2M HOAc, pH 5.5, 350 g/l GuHCl guanidinium HCl

PB Buffer: 5 M guanidine hydrochloride, 30% isopropanol.

NaI Buffer: 6 M NaI,  stored in darkness, Sigma, 21763, expensive

PE Buffer:

75% EtOH.
25 mM NaCl,
5 mM Tris-HCl, pH 7.5

1. Inoculate 4~5 ml culture medium with a single E. coli colony containing appropriate plasmids. Grow overnight at 37° C. with vigorous shaking;

2. Spin down the bacteria at 3000 ~ 6000 rpm for 5 min. Discard the supernatant;

3. Add 250 µl of SI into the tubes. Vortex to re-suspend the pellet;

4. Add 250 µl of SII, mix to lyse the bacteria;

5. Add 350 µl of SIII  or N3 to the tube. Mix by shaking.

6. Pour liquid to centrifuge tubes, put tubes on dry ice for 1 min.

7. Spin for  2.5 min for two times. Transfer the supernatant to a spin column.

8. Wash column with 0.35 ml of PB or NaI buffer, spin, remove liquid

9. Wash column with 2 X 0.35 ml of PE, spin, remove liquid

10. Spin at 13000 rpm for 1 min.

11. Add 50 µl water or TE or EB to elute DNA into a new tube.

Silica column:

1. Add 5 g silicon dioxide (Sigma, S5631) to 50 ml sterile water in a 50 ml Falcon tube

2. Settle for 2 h.
3. Remove supernatant (containing fine silica particles with sizes below ca. 1 ÎŒm)

4. Resuspend the pellet in 50 ml sterile water

5. Let settle for another 2 h.

6. Discard supernatant.

7. Resuspend silica in 50 ml sterile water to make a  final concentration of approximately 100 mg/ml.
The slurry can be stored at room temperature and will be stable for at least 12 months.

One mg of the silicon dioxide can bind 3-4.5 ÎŒg DNA.
8. Add Add 20 ÎŒl silica matrix to a spin column.