Plasmid MaxiPrep by Ca Q Sepharose FF
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 CalCl2), 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:
[edit]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)