Screnning colony by restriction digestion

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

5% (w/v) CTAB
Cetyl trimethylammonium bromide

1. Inoculate 4 ml LB or 2XYT culture medium (containing antibiotics) with a single E. coli colony containing appropriate plasmids. Grow overnight at 37 oC with vigorous shaking;

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

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

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

5. 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.

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

7.  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 1.7 or 2 ml tube

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

9. 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

10. Add 0.5 ml of 70% EtOH or PE

11. Spin 1 minute. Discard liquid,  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)

12. 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).

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. 

16. Wash with 700 ul PE, spin.

17. Spin 1 min

18. Add 55 ul EB to elute plasmid DNA

19. Use 10 ul for sequencing.

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

21. Label plasmid clone corresponding sequencing verified clone, store at -20 oC