Editing Plasmid MaxiPrep Q Membrane (section)

== &nbsp; Gel Filtration + Q membrane  ==

1. Grow high copy number plasmid in DH5alpha in 1 L LB/Amp or LB/Kan o/n at 37oC. 

2. Spin down cells, <br> 

3. Resuspend cells with cold 10 ml to 20 ml P1 buffer/per gram of pellet, if less than 2 g, use 10 ml of P1.&nbsp; Use a stainless steel spatula and votex to break big pellet into homogeneous supension.<br> 

4. Lyse cells with&nbsp; 10 ml to 20 ml of P2/per gram of pellet. Mix gently until cells are completely lysed. Incubate at RT for 5 min to digest RNA.<br> 

5. Neutralize with cold 10 ml to 20 ml of P3/per gram of pellet. Mix gently in the begining, then votex a few seconds.<br> 

6. If P3 without acetic acid or ammonium acetate is used, you can incubate the suspension 4 hours to o/n to precipitate RNA.

7. Spin for 5 min at 6000 rpm, Transfer supernatant to 50 ml tubes, Spin at 12000 rpm for 10 min using high speed centrifuge, collect supernatant, filter liquid using a coffee filter paper. <br> 

8. Add 0.7 volume of isopropanol, incubate on ice for 5~15 min. Do not inubate for too long as protein and other junk will co-precipitate with DNA/RNA. Spin at 12000 rpm for 15 min. 

9. Wash pellet by spraying with 1 ~3 ml 70% ethanol. Drain liquid each time. Put tube on top of paper towel for a few minutes.&nbsp; Resuspend pellet in 4 ml (Total &lt;4.5 ml) Buffer A or TE. Pellet may not be soluble, so use 1 ml filter pipette tip to disrupt the pellet into smaller pieces. 

10. Add 10~15 ul of 50 mg/ml RNAse A, incubate at 37 oC for 1 hour or RT for o/n. The insoluble RNA/protein/DNA pellet should have been dissolved. Hopefully most of the RNA should be degraded.<br> 

11. Transfer&nbsp; ~1.3 ml to 1.5 ml microcentrifuge tubes. Label them! Be careful not to cross contaminate or knock-over.<br> 

12. Add ~75 ul of 10% Triton X-114 to each tube (do not need to be accurate, do not need to change tip, do not corss contaminate or knock-over, just add to the top of tubes, do not touch the inside of tubes), close the lids, mix for a few seconds by votexing, incubate on ice for 3 min.<br> 

13. Heat at 55 oC for 5 min.<br> 

14. Spin at 13000 rpm for 3 min.<br> 

15. Transfer supernatant to a clean tube. DO NOT take white interface (protein) and bottom phase.<br> 

16. Load &lt;=5 ml to a BioRad (Bio-Sil TSK250) (Tosoh) HPLC gel filtration column, separate RNA from DNA using program 47,&nbsp; filtered Buffer D (10 mM Tris-HCl, pH7.5, 1 mM EDTA, 50 mM NaCl). Flow rate: 2.5 ml/min, 4 ml per fraction. DNA should come out at fraction 21. <br> 

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Be very careful to avoid cross contamination, before injection of plasmid solution: 

*Wash the enternal injection valve extensively with clean ddH2O 
*Wash injection syringe at least 5 times. 
*Wash injection loop at least 5 times

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17. Combine the first peak fractions containing plasmid DNA. 

Wash glass tubes immediately with ddH2O at least 4 times. Dry them top-down to avoid dust. 

After FPLC, wash injection valve, loop, syringe and needle 5 times with water. 

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18. Activate Q membrane with 4 ml of 1 M NaOH then 10 ml of 2 M NaCl. 

19. Equilibrate Q membrane with '''Buffer A''' (0.5 M NaCl in 10 mM Tris buffer, 1 mM EDTA, pH 8.0.)<br> 

'''SartoBind Q:''' Dissolve DNA in &amp; washing with '''0.5 M''' NaCl in 10 mM Tris buffer, 1 mM EDTA, pH 8.0. <br> 

20. Load&nbsp; DNA solution to one equilibrated <strike>Mustung</strike> '''SartoBind''' Q membrane disk using a syringe of peristaltic pump at&nbsp; flow rate of 1 ~ 4 ml/min.<br> 

21. Wash disk with '''5''' ml of '''Buffer B '''(25 mM Tris-HCl, '''0.5''' M NaCl, '''10% EtOH, 0.5% Triton X-114''', 1 mM EDTA, pH 8.0). <br> 

This wash contains Triton X-114, which has high UV absorbance, so there is no need to measure OD260.<br> 

This wash will elute endotoxin and residual RNA.<br> 

22. Wash with 20 ml of '''Buffer A'''&nbsp; (0.5 M NaCl, 10 mM Tris buffer and 1 mM EDTA at pH 8).<br> 

This wash is to wash out Triton X-114 and residual RNA<br> 

23. Elute plasmid DNA slowly using 15~ 20 ml of '''Buffer C''' ('''1.3 M''' NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA), Discard the first 1 ml, Soak Q membrane in this buffer for 3 min, then slowly (0.5 ml/min) elute DNA using peristaltic pump. Collect elute in 50 ml tube. <br> 

'''SartoBind Q:''' Clean Q membrane with 5 ml of 1 M NaOH, then&nbsp; 10 ml of 2.5 M NaCl.&nbsp; Wash Q membrane with 10 ml water. Do not discard the membrane. It is about $250 each.<br> 

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24. Measure DNA concentration using nanodrop.<br> 

25. Precipitate DNA using 0.7 vol (14 ml) of isopropanol,&nbsp; incubation on ice for 15 to 30 min.<br> 

26. Spin down DNA using high speed centrifuge 12000 rpm for 15 min.<br> 

'''Inside a biosafety hood:'''<br> 

27. Wash 2X with 2 ml 70% ethanol (by spray ~2 to 4 ml of 70% ethanol). Decant enthanol.<br> 

28. Dissolve DNA in 5 X 200 ul TE<br> 

29. Measure DNA concentration and OD260/280 ratio, check DNA quality by agarose gel electrophoresis.<br>