Editing Plasmid MaxiPrep by CTAB Q Sepharose FF (section)

== Protocol 1<br>  ==

1. Inoculate 500 mL 2xYT or LB/Amp or Kan from 1-5 mL overnight 

2. Grow at 37 ºC overnight. 

3. Spin cells at 6000 rpm for 10 min at 4ºC using GS3 rotor. 

4. Resuspend cells in 8 mL P1. 

5. Add 8 mL P2. Stir very gently using a long 5 ml plastic pippette, until homogenous and clear. Put on ice for 10 min. 

6. Add 10 mL P3 (3 M KAc, pH 5), or S3 (Qiagen), mix, put on ice for 10 min. S3 is the best. 

7. Spin 6000 rpm at 4ºC for 10 min. 

8. Filter supernatant using autoclaved cheesecloth or coffee filter paper. 

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Optional steps 

9. Add 3% CTAB solution to supernatant to final concentration of 0.3%.&nbsp; <strike>0.18%</strike> 

10. Spin down CTAB-DNA complex. Dissolve pellet in 0.7 M NaCl or 3M KAc (Potassium Acetate) 

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11. Load DNA solution to Q Sepharose FF column or HiTrap Q FF column (GE Healthcare) pre-equilibrated with QBT 

12. Wash column with QC buffer (0.27~0.45M NaCl). 

13. Elute DNA with QF buffer. 

14. Precipitate DNA using 0.6 vol of isopropanol or 2 vol of ethanol. 

15. Wash DNA precipitate with 75% ethanol. 

16. Dry DNA in biosafety hood. 

17. Dissolve DNA in water or TE. 

18. Analyse DNA purity by agarose gel and nanodrop. 

'''Regenerate Q column:''' 

19. Add 1 mg/mL DNase I, 50mM Tris-HCl, 10mM MgCl2, 250mM NaCl, pH 7.6 and put column or resin in 37 oC incubator over night. 

<br>20. Wash with:<br>5CV 1M NaOH<br>5CV H<sub>2</sub>O<br>5 CV isopropanol<br>5 CV water<br>5 CV 1M phosphoric acid<br>5 CV water<br>5 CV 2M NaCl.<br>5 CV 0.35 M NaCl in 20% ethanol. 

'''Buffer QBT''' 

Dissolve 43.83 g NaCl, 10.46 g MOPS (free acid) in 800 ml dH<sub>2</sub>O. Adjust the pH to 7.0 with NaOH. Add 150 ml pure isopropanol and 15 ml 10% Triton X-100 solution (v/v). Adjust the volume to 1 liter with dH<sub>2</sub>O. 

750 mM NaCl<br>50 mM MOPS pH 7.0<br>15% isopropanol (v/v)<br>0.15% Triton® X-100 (v/v) 

'''Buffer QC''' 

Dissolve 58.44 g NaCl and 10.46 g MOPS (free acid) in 800 ml dH<sub>2</sub>O. Adjust the pH to 7.0 with NaOH. Add 150 ml pure isopropanol. Adjust the volume to 1 liter with dH2O. 

1.0 M NaCl<br>50 mM MOPS pH 7.0<br>15% isopropanol (v/v) 

<br>'''Buffer QF''' 

Dissolve 73.05 g NaCl and 6.06 g Tris base in 800 ml dH2O and adjust the pH to 8.5 with HCl. Add 150 ml pure isopropanol. Adjust the volume to 1 liter with dH<sub>2</sub>O. 

1.25 M NaCl<br>50 mM Tris·Cl pH 8.5<br>15% isopropanol (v/v)<br><br>