Editing Plasmid MaxiPrep by PEG Q XL (section)

== Protocol 2: HiTrap 1 ml or 5 ml QXL column<br>  ==

1. Grow high copy number plasmid in DH5alpha in 500 ml or 1L LB/amp or LB/Kan o/n at 37oC.<br> 

2. Spin down cells<br> 

3. Resuspend cells with 20 ml P1 buffer (no RNase).<br> 

4. Lyse cells with 20 ml of P2.<br> 

5. Neutralize with 20 ml of P3.<br> 

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

7. Spin at 12000 rpm for 10 min, high speed, collect supernatant, filter liquid using coffee filter is necessary.<br> 

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8. Add 20 ml 40% PEG 8000 to&nbsp; supernatant (1: 4 v/v)<br> 

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''Alternatively,'' the DNA in supernatant from step 7 can be precipitated first with isopropanol, dissolve DNA in 1 ml of water, add 0.25 ml of 4 M NaCl (final is 0.8 M), add 0.25 ml of 40% PEG8000 (final is 6.5%) and spin down DNA using a benchtop centrifuge.<br> 

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9. Spin for 10 min at 25000 rpm, ultracentrifuge. <br> 

10. Resuspend pellet in 1 to 5 ml&nbsp; '''water''', aliquote to 1.5 ml tubes.<br> 

11. Add '''X ul of 10% Triton X-114 (10X) '''to the plasmid DNA solution to adjust the final concentration of TritonX-114 to&nbsp;'''1%''' '''(1:10).''' Mix by votexing. 

12. Incubate '''on ice for 10 min'''.<br> 

13. Incubate at 42~55 oC for 10 min 

14. Spin at 13000 rpm (microcentrifuge) for 5 min. Collect and combine '''supernatant''' to a new tube,&nbsp; chill on ice. Discard the tubes containing bottom phase.<br><br> 

15. Equilibrated Q XL column with '''QBT '''or 5 CV of&nbsp; 0.5M KAc (potassium acetate), pH5.5 

16. Load 1~5 ml '''Supernatant '''(containing plasmid DNA) slowly to '''1 ml Q XL column''' using a syringe.<br> 

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Note: if a large volume of DNA sample or supernatant is to be applied, use a peristaltic pump at a flow rate of 1 ml/min to load sample.<br> 

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17. Wash with 3 ml of 25 mM Tris-HCl, 0.75 M NaCl, <strike>'''1% Triton X-100 or 1% Triton X-114,'''</strike> 1 mM EDTA, pH 8.0 by pushing liquid '''<u>slowly</u> '''to the column using&nbsp; a syringe.<br> 

<strike>18. Wash column with 25 mM Tris-HCl, 1 mM EDTA, pH 8.0 until Triton X-114&nbsp; is completely washed off column.</strike><br>18. Wash with 5 ml of 25 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA, pH 8.0 by pushing liquid '''slowly''' to the column using a syringe.<br>19. Elute DNA with 3 ml of 50 mM Tris-HCl,&nbsp; 1 M NaCl, pH 8.0 by pushing liquid '''slowly''' to the column using a syringe.<br> 

(after elution, clean the column using cleaning solution: 5 ml of 0.5 M NaOH, 2 M NaCl using a syringe).<br> 

20. Combine fractions containing DNA.<br> 

21. Add 0.7 volumes&nbsp; of isopropanol, &nbsp;(optionally add 3M potassium acetate, 10% final volume).<br> 

22. <font color="red">Aliquot to 1.5 ml Eppendorf tubes</font>, Spin at 13000 rpm for 15 min using a benchtop centrifuge to precipitate DNA.<br> 

23. Add 300 ul of 80% ethanol gently, spin briefly.<br> 

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'''Step 24 and 25 should be peformed inside a biosafety hood'''<br> 

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24. Remove ethanol, and then dry inside a biosafety hood.<br> 

25. Add 0.3 ml sterilized TE to dissolve DNA, add 3 X 100 ul to wash DNA off the tubes. Combine and take 5 ul out to another tube.<br> 

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26. Measure DNA concentration and purity using a Nanodrop; Run gel to check RNA level; Digested with restriction enzymes to confirm identity.<br> 

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

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