Paul.xu at 20:59, 5 October 2011

2011-10-05T20:59:48Z

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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 8 mL '''P3-Ca''' (3 M KAc, pH 5.0, '''1.2 M CalCl''''''2'''), mix, put on ice for 10 min.

7. Spin 6000 rpm at 4ºC for 10 min.

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

9. Load supernatant to Q Sepharose FF column or HiTrap Q FF column (GE Healthcare) pre-equilibrated with QBT

10. Wash column with QC buffer (0.27â0.45M NaCl).

11. Elute DNA with QF buffer.

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

13. Wash DNA precipitate with 75% ethanol.

14. Dry DNA in biosafety hood.

15. Dissolve DNA in water or TE.

16. Analyse DNA purity by agarose gel and nanodrop.

== Regenerate Q column: ==

17. Add 1 mg/mL DNase I, 50mM TrisâHCl, 10mM MgCl2, 250mM NaCl, pH 7.6 and put column or resin in 37 âŠC incubator over night. 18. Wash with: 5CV 1M NaOH 5CV H2O 5 CV isopropanol 5 CV water 5 CV 1M phosphoric acid 5 CV water 5 CV 2M NaCl. 5 CV 0.35 M NaCl in 20% ethanol.

'''P3-Ca'''

3 M KAc, pH 5.0 1.2 M CalCl2

 '''Buffer QBT'''

Dissolve 43.83 g NaCl, 10.46 g MOPS (free acid) in 800 ml dH2O. 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 dH2O.

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

'''Buffer QC'''

Dissolve 58.44 g NaCl and 10.46 g MOPS (free acid) in 800 ml dH2O. 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 50 mM MOPS pH 7.0 15% isopropanol (v/v)

 '''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 dH2O.

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

[[Category:Protocols]]

Plasmid MaxiPrep by Ca Q Sepharose FF - Revision history

Paul.xu at 20:59, 5 October 2011