Paul.xu at 17:55, 25 June 2017

2017-06-25T17:55:23Z

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'''Mini-prep using PEG 8000'''

'''S I''' 50 mM Tris-HCl, 10 mM EDTA, pH 8.0

Optional: add 1 ml of 100 mg/ml thymolphthalein ('''lyseblue''') in ethanol to 100 ml S I solution

Note: This S1 solution does not contain RNAse A.

'''S II'''

0.2M NaOH and 1% SDS; 8 g NaOH, 50 ml 20% SDS(w/v), dilute to 1 L

'''S III'''

2.0M HAc, 0.6M KAc, 0.4M MnCl2 , 0.4 M CaCl2;

'''40% PEG 8000'''

 <strike>'''5% (w/v) CTAB''' Cetyl trimethylammonium bromide</strike>

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

2. Spin down bacteria culture 13000 rpm for 30 sec (microcentrifuge), or 3000 rpm for 5 min (big centrifuge). Discard the supernatant;

3. Add 100 µl of  S1  or  P1 or GTE solution into the tubes. Vortex to re-suspend the pellet; Transfer liquid to a microcentrifuge tube

4. Add  200 µl  P2 (0.2 N NaOH/1% SDS )solution to the bacterial suspension. Mix by inverting/swirling. Do not vortex. Place on ice for 5 minutes.

5. Add  200 µl of N3 or P3 (3 M potassium acetate, pH 5) or S3. Immediately vortex for exactly 3 seconds to mix.   

<strike>'''Optional:''' add 5% (w/v) CTAB to '''0.18%''' final concentration, incubate '''15''' min at room temperature.</strike>

6.  Cool the mixture with dry ice for 1 min, Spin in a  centrifug at full speed (13000 rpm) for 4 min to pellet cell debris and chromosomal DNA. . Pour the supernatant to a new 2 ml tube;

7. Add 2 volumes (0.8 ml) of 100 % ethanol, or 1 volume (0.5 ml) of isopropanol . Vortex to mix well. Hold at room temperature 5 minute

8. Spin 10 minute at room temperature in microfuge to pellet nucleic acids. Discard liquid by pouring, and the tubes are drained upside down on a paper towel

9. Add 0.5 ml of 70% EtOH or PE

10. Spin 1 minute. Discard liquid.   and the tubes are drained upside down on a paper towel.  Use 10 ul pippet tip to remove residual liquid. (opposite side of the white DNA/RNA pellet)

11. Resuspend pellet in  50 µl TE, pH 8.0 + 10 µg/ml RNase A (add 1 µl  of 10 mg/ml RNase A into 1 ml TE).

Optionally:  digest  for 30 minutes at 37°C to degrade RNA

Immediately, use 5 µl of resuspended DNA/RNA for restriction digest.

== PEG purification ==

1. Chill on ice. Microfuge for 10 minutes at top speed at 4°C. 

2. Transfer supernatant to fresh tube.

3. Add an equal volume of sterile 1.6 M NaCl + 13% (w/v) PEG 8000 + 20 mM MgCl2. Vortex well to mix completely.

'''PEG Solution''': 20 ml of 4 M NaCl, 16.3 ml 40% PEG8000, 1 ml 1 M MgCl2, H2O to 50 ml

4. Hold on ice 1 hour to overnight. Overnight will give ~100% yield; 1 hour will give ~50% yield.

5. Spin for 30 minutes at top speed at 4°C. Remove supernatant carefully. Pellet may be small.

6. Add 0.5 ml 70% EtOH and vortex briefly.

7. Spin in microfuge for 5 minutes at top speed at 25°C. Remove 70% EtOH.

8. Speed-Vac for ~5 minutes to dry pellet. Resuspend pellet in original volume of TE buffer

PEG-Plasmid-Mini-Prep - Revision history

Paul.xu at 17:55, 25 June 2017