Editing Plasmid MaxiPrep Silica Oxide (section)

== MaxiPrep  ==

1. Inoculate 1 L mL 2xYT or LB/AMP or Kan from 1-5 mL overnight<br>2. Grow at 37 oc overnight. <br>3. Spin cells at 6000 rpm for 10 min at 4ºC using GS3 rotor.<br>4. Resuspend cells in 20 mL P1 without RNase A.<br>5. Add 20 mL P2. Stir very gently using a long 5 ml plastic pippette, until homogenous and clear.<br>6. Add 20 ml P3, mix, add 100 mg/ml RNAse sol to 0.1 mg/ml. Leave it on bench to digest o/n<br>

7. Spin 6000 rpm at 14ºC for 10 min.<br>8. Filter supernatant using a coffee filter paper.<br>

9. Add 0.5 g dry weight equivalent of SiO2 (5 ml of 100 mg/ml)<br>10. Add 0.6 volume isopropanol, 3.3% CTAB to final conc 0.1%. Mix. Leave it at room-temperature for 15 min to 1 hour.<br>11. Spin at 5000 rpm at 25 ºC for 10 min. <br>12. Resuspend pellet in 10 ml PB, transfer to 50 ml clean centrifuge tube<br>

13. Spin 1 min at 5000 rpm.<br>

14. Wash again with 5 ml PB.<br>

15. Wash 2X with 5 to 10 ml PE, resuspend everytime time<br>

16. Add 1.5 ml preheated to 80 oC TE to suspend. Incunate in 80 oC water for 5 min<br>

17. Spin and collect eluted DNA, transfer liquid containing DNA to a 1.5 ml tube for storage.<br>

18. Add another hot 1.5 ml to elute again. Check OD260 to see if DNA is completely eluted.<br>19. Measure DNA concentration and run 5 ul on agarose gel to check for the presence of RNA. 

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The problem of this method is: too difficult to get rid of RNA<br>

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<br>'''P3 (~5.5) [500 mL]'''<br>147g KOAc (3M)<br>55 mL glacial HOAc<br>'''PE:''' 20 mM Tris-Cl pH 7.5, 2 mM EDTA, 0.4 M NaCl, and 50% v/v ethanol<br>