Editing Plasmid MaxiPrep by PEG Q XL (section)

== Protocol 1: 1 L culture with ~3 to 5 ml QXL column  ==

For 4 L culture a column packed with 10 ml QXL resinshould be used.<br> 

For 1 L culture, the column can be packed with 3 to 5 ml of QXL resin. 

1. Grow high copy number plasmid in DH5alpha in 1 L LB/Amp or LB/Kan o/n at 37<sup>o</sup>C. 

2. Spin down cells 

3. Resuspend cells with '''20''' ml P1 buffer (no RNase). 

4. Lyse cells with '''20 '''ml of P2. 

5. Neutralize with '''20''' ml of P3. 

6. Add&nbsp;'''15''' ml of '''5 M''' CaCl<sub>2</sub> to final conc of 1 M, cool on ice for 5 min. 

7. Spin at 12000 rpm for 10 min, high speed centrifuge, collect supernatant, filter liquid using coffee filter if necessary. 

8. Add '''20''' ml of&nbsp; 40% PEG 8000 to&nbsp; supernatant (1:4, v/v). Transfer to ultracentrifuge tubes, balance to 0.1 g. Number tubes with small piece of adhesive tape. Put into a 35K rotor.<br>9. Spin for 15 min at 25000 rpm, 20 oC using ultracentrifuge. <br> 

10. Make sure you know where is the pellet as it is transparent. Resuspend "pellet" in 0.8 ml water, wash 5 times with water, transfer to a clean tube. Measure DNA concentration using Nanodrop. Calculate yield.<br> 

11. Add X ul of 10% Triton X-114 (10X) to the plasmid DNA solution to adjust the final concentration of Triton X-114 to 1% (1:10). Mix by votexing. Put on ice. 

12. Incubate on ice for 5 to 10 min. You will see the solution is clear.&nbsp; Aliquot to 1.5 ml tubes.<br> 

13. Incubate at 42~55 <sup>o</sup>C for 5 to 10 min. You will see the solution is turbid. 

14. Spin at 13000 rpm (microcentrifuge, max speed) for 2.5 to 5 min. Carefully collect and combine supernatant to a new tube (Do not take any button phase). Discard the tubes containing bottom phase. 

15. Equilibrate 3 to 5 ml Q XL column with QBT or 5 CV of 0.5M KAc (potassium acetate), pH 5.5. 

16. Load DNA solution from step 14 to&nbsp;Q XL column, collect FT. The OD260 should be very low (3 ng/ul or none) in FT.<br>17. Wash with 3 CV (e.g., 3 X3 ml) of 25 mM Tris-HCl, 0.75 M NaCl, 1 mM EDTA, pH 8.0. Collect wash. OD260 should be low. 

18. Wash with 5 CV (e.g., 3X 5 ml) of 25 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA, pH 8.0. Collect wash.&nbsp; DO 260 should be low.<br>19. Elute DNA with 5 CV (e.g., 15 ml) of 50 mM Tris-HCl, 1 M NaCl, pH 8.0. Collect eluate. 

(after elution, clean the column using cleaning solution: 15 ml of 0.5 M NaOH, 2 M NaCl, wash column with water and soak the resin in 20% ethanol until next use. The beads should be stored at 4 oC for long term storage). 

20. If fractions are collected, measure each fraction with Nanodrop and combine fractions containing DNA. 

21. Add equal volume of&nbsp; isopropanol, (optionally add 3M potassium acetate, 10% final volume). Transfer to ultracentrifuge tube, balance the tubes with isopropanol.<br> 

22. Spin at 25000 rpm, 20 oC for 15 min to precipitate DNA. <br> 

'''Step 23 and 26 should be peformed inside a biosafety hood''' 

23. Take centrifuge tubes to the biosafety hood. Discard liquid careful, make sure you know where is the pellet as it is invisible (transparent). 

24. Spray some&nbsp; 80% ethanol into the tube gently, do not disturb pellet. <br> 

25. Remove ethanol gently,&nbsp; dry pellet for 5 min inside a biosafety hood. 

26. Add 200 ul sterilized TE to dissolve DNA (you will not see pellet because it is transparent), add 3 X 200 ul to wash DNA off the tubes. Combine and take 10 ul out to an Eppendorf tube. 

26. Take 2 ul to measure DNA concentration and purity using a Nanodrop; Run 2 ul on gel to check RNA level; Digest with restriction enzymes and run on agarose gel to confirm identity. 

27. Optional: Analyse endotoxin level to see if it is acceptable for transfection. 

Yield: should be ~1 mg/L culture<br>Quality: RNA &lt;1%, 260/280: &gt;1.8, mostly supercoil DNA<br>