Paul.xu at 22:06, 3 August 2011

2011-08-03T22:06:50Z

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'''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. 

 

 

[[Category:Protocols]]

Improved-Home-Made-Plasmid-Mini-Prep - Revision history

Paul.xu at 22:06, 3 August 2011