Editing Plasmid MaxiPrep HIC (section)

=== Hexyl 650C (Toso) ===

Hexyl 650C can be used to remove endotoxin at 2.2M ammonium sulfate. Plasmid will not bind to the resin.

at 3 M ammonium sulfate plasmid will bind to resin and can be eluted using 1 M ammonium sulfate.



1. Spin down 500 ml cells

2. Add 20 ml of P1 (50 mM Tris-HCl, 10 mM EDTA pH8.0) without RNase.

3. Add 20 ml of P2 (200 mm NaOH, 1% SDS solution), lyse for about 5 min.

4. Add 20 ml of P3 (3 M potassium acetate, pH ~ 5. Dissolve 294.5 g potassium acetate in 500 ml dH2O. Adjust the pH to 5.5 with ~110 ml glacial acetic acid and bring to 1 L with ddH2O).

5. Add 15 ml of 5 M CaCl2. Final conc is 1 M. (This step should be ommitted if P1 has RNase)

1*(60+X)=5*X; (5X-1X)=60; 4X=60;

5. Put on ice for 10 min, spin at 6000 rpm for 10 min. Filter floating stuff with coffer filter.

6. Collect supernatant, add isopropanol to final 70% (adding 2.1 ml isopropanol per 3 ml of clarified lysate).&nbsp;

7. Spin.

8. Wash pellet with 70% ethanol.

9. Dry and dissolve DNA in TE buffer (10 mM Tris-HCl, 0.1mM EDTA, pH 8.0.) or water, The DNA solution can be frozen at -20.degree. C. until use

10. Pack a Butyl 650S or 650M column with a bed volume of 3 to 5 ml <strike>(1.6 cm X 10 cm).</strike>

11. Equilibrate column with 2 CV of&nbsp; 3 M ammonium sulfate (AS) in 50mM Tris-HCl, 10 mM EDTA (TE) buffer, pH 7.4

12.&nbsp; Load solution to column at 1 ml/min

13. Wash column with 15&nbsp; to 20&nbsp; ml of&nbsp; 3 M AS in TE buffer, then 15 ml of 80% Ethanol or PE.

14. Elute DNA with 2 ml&nbsp; of TE or water. (Alternatively collect 1 ml fractions). Discard the first 2 ml.

15. Measure DNA concentration and 260/280 ratio in each fraction. Combine fractions containing DNA.

16. If purity is not good, purify further using Q XL resin.

<strike>1. Dissolve pellet in 20mM KPB (potassium phosphate buffer, 20 mM potassium phosphate monobasicsolution combined with 20 mM potassium phosphate dibasic in a proportion to obtain a pH of 6.8), pH 6.8 and 2M ammonium sulfate (AS).</strike>

<strike>2. Load solution to 1.6 cm X 4 cm (8 ml cv) Hexyl 650C column equilibrated with KPB, pH 6.8, containing 2M AS, at a flow rate of 2 ml/min.</strike>

<strike>3. Wash column with KPB containing 2M AS.</strike>

<strike>4. Elute DNA with TE.</strike>

<strike>5. Clean column with 0.5N NaOH.</strike>

<strike>6. Wash column with 15 CV of ddH2O.</strike>