Editing PEG-Plasmid-Mini-Prep

<u>'''Mini-prep using PEG 8000'''</u>

'''S I'''<br/>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

<u>Note: This S1 solution does not contain RNAse A.</u>

'''S II'''

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

'''S III'''

<u>2.0M HAc, 0.6M KAc, 0.4M MnCl2 , 0.4 M CaCl2;</u>

'''40% PEG 8000'''

<br/><strike>'''5% (w/v) CTAB'''<br/>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 &nbsp;S1 &nbsp;or &nbsp;P1 or GTE solution&nbsp;into the tubes. Vortex to re-suspend the pellet;&nbsp;Transfer liquid to a microcentrifuge tube

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

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

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

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

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

8. Spin 10&nbsp;minute at room temperature in microfuge to pellet nucleic acids. Discard liquid by pouring, <span style="color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif; font-size: small;">and the tubes are&nbsp;drained upside down on a&nbsp;</span><span style="font-weight: bold; color: rgb(106, 106, 106); font-family: Roboto, arial, sans-serif; font-size: small;">paper towel</span>

9. Add 0.5 ml of 70% EtOH or PE

10. Spin 1 minute. Discard liquid. &nbsp;&nbsp;<span style="font-size: small; color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif;">and the tubes are&nbsp;drained upside down on a&nbsp;</span><span style="font-size: small; font-weight: bold; color: rgb(106, 106, 106); font-family: Roboto, arial, sans-serif;">paper towel.&nbsp;</span> Use 10 ul pippet tip to remove residual liquid. (opposite side of the white DNA/RNA pellet)

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

Optionally: &nbsp;digest &nbsp;for 30 minutes at 37°C&nbsp;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.&nbsp;

2.&nbsp;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