Editing Screnning colony by restriction digestion

<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 LB or 2XYT culture medium (containing antibiotics)&nbsp;with a single E. coli colony containing appropriate plasmids. Grow overnight at 37 oC with vigorous shaking;

2. Spin down bacteria culture at&nbsp;3000 rpm for 5 min (big centrifuge). Discard the supernatant;

3. Add 100 µl of &nbsp;S1 &nbsp;solution&nbsp;into the tubes. Vortex to re-suspend the pellet;&nbsp;Transfer liquid to &nbsp;microcentrifuge tubes using 200 ul pipette

4. Add 1 ml &nbsp;LB or 2XYT&nbsp;culture medium&nbsp;containing antibiotics&nbsp; back to each culture tube. Grow in shaker incubator at 37 oC.

5. 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.

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

7. &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 1.7 or 2 ml tube

8. Add&nbsp;2 volumes (1&nbsp;ml) of 100&nbsp;% ethanol, or 1 volume (0.5 ml) of isopropanol . Vortex to mix well.&nbsp;

9. Spin 10&nbsp;minute at room temperature in microfuge to pellet nucleic acids. Discard liquid by pouring,&nbsp;<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>

10. Add 0.5 ml of 70% EtOH or PE

11. Spin 1 minute. Discard liquid,&nbsp;<span style="font-size: small; color: rgb(84, 84, 84); font-family: Roboto, arial, sans-serif;">&nbsp;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>&nbsp;Use 10 ul pippet tip to remove residual liquid. (opposite side of the white DNA/RNA pellet)

12. 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).

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

14. For positive clones, further purify plasmid by adding 500 ul PB buffer to the above DNA/RNA solution and transfer liquid to silica spin column

15. Spin 30 sec.&nbsp;

16. Wash with 700 ul PE, spin.

17. Spin 1 min

18. Add 55 ul EB to elute plasmid DNA

19. Use&nbsp;10 ul for sequencing.

20 After overnight culture, add 0.75 ml 80% glycerol to&nbsp;corresponding sequencing verified clone, transfer e coli&nbsp;to a clean tube, print lable and store at -80C

21. Label plasmid clone&nbsp;corresponding sequencing verified clone, store at -20 oC
[[Category:Protocols]]