Paul.xu at 15:52, 16 October 2012

2012-10-16T15:52:47Z

New page

Resin is too expensive

== Purification of Plasmid using PlasmidSelect Xtra Resin ==

 

For 1 L culture, the column can be packed with  5 ml of PlasmidSelect resin.

1. Grow high copy number plasmid in DH5alpha in 1 L LB/Amp or LB/Kan o/n at 37oC.

2. Spin down cells

3. Resuspend cells with 20 ml P1 buffer (no RNase).

4. Lyse cells with 20 ml of P2.

5. Neutralize with 20 ml of P3.

6. Spin at 12000 rpm for 10 min, high speed centrifuge, collect supernatant, filter liquid using coffee filter if necessary.

7. Load clarified solution to Sepharose 6 Fast Flow XK 50/30 = 20 cm bed height, 10 ml/min (30 cm/h). Prequlibrated with Buffer A. 

Load up to 130 ml or 0.3 column volumes (CV) 

 '''Buffer A''': 2.0 M Ammonium sulphate, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0 

Wash column with Buffer A and Collect '''void fraction''' containing DNA, RNA is eluted at ~0.7 CV.  Wash column until no protein and DNA can be detected in eluate. 

8. Load solution from step 7 (void fraction) to PlasmidSelect XK 16/20 = 15 cm bed height, 4 ml/min (120 cm/h), equilibrated with Buffer A. 

Wash with 10 CV of  Buffer A 

 

Elute DNA isocratically with  5 CV Buffer B. 

 '''Buffer B''': 1.4 M NaCl, 2.0 M Ammonium sulphate, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0. 

Pooled elution peak, diluted with '''four''' volumes of water (so the NaCl concentration is 0.35 M). 

7. Polishing and concentration 

Load diluted DNA solution to a SOURCE 30Q XK 16/20 = 15 cm bed height, 4 ml/min (120 cm/h) equilibrated with Buffer C 

 '''Buffer C''': 0.4 M NaCl, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0 '''Buffer D''': 1.0 M NaCl, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0, 

gradient from 0–100% D in 5 CV 

Or wash with 0.4 M NaCl for 2 CV, and elute with  Buffer D. 

 

 

8. After elution, clean the columns using cleaning solution (CIP, Cleaning-in-place): 2 CV of 0.5 M NaOH, 2 M NaCl, wash columns with water and soak the resin in 20% ethanol until next use. The beads should be stored at 4 oC for long term storage).

 

9. If fractions are collected, measure each fraction with Nanodrop and combine fractions containing DNA.

10. Add equal volume of isopropanol, (optionally add 3M potassium acetate, i.e. P3, 10% final volume). Transfer to ultracentrifuge tube, balance the tubes with isopropanol.

10. Spin at 25000 rpm, 20 oC for 15 min to precipitate DNA. 

11. Rinse with 80% ethanol. Dry in biosafety hood. Dissolve DNA in endotoxin-free TE or water. Take 5 ul to a clean tube for analysis by nanodrop and agarose gel. 

 

12. If P1 with RNase is used, Step 7 maybe omitted. Dilute clarified alkaline lysate 1.5 volume with  Buffer E,

'''Buffer E:''' 3.75 M (NH4)2SO4, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0.

Load sample on a  column packed with PlasmidSelect. Wash column with  Buffer A.

Elute DNA with Buffer F at 120 cm/h for 5 CV. 

'''Buffer F''': 3M NaCl, 2M (NH4)2SO4, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0 

Wash column with water. RNA and other contaminants such as proteins and endotoxins will be eluted by water.

Finish purification as step 7- 11.

 

== One Step Purification of Plasmid DNA ==

1. Dilute lysate with 1.1 volume of 4.0 M (NH4)2SO4, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0. Final (NH4)2SO4  concentration is 2.2 M 

2. Load to a long column packed with PlasmidSelect.

3. Elute DNA with  3 M NaCl in 2.25 M (NH4)2SO4, 10 mM EDTA, 100 mM Tris-HCl, pH 7.0 at 120 cm/h for  5 CV.

4. Wash column  water to elute RNA. 

[[Image:Plasmid select.jpg]]

== Remove Endotoxin and open-circle plasmid DNA from supercoiled plasmid using PlasmidSelect Xtra ==

The binding capacity of PlasmidSelect Xtra resin is 1 mg DNA per ml resin.

 

Lysate should be prepared using P1 + RNase, P2, P3; 

Plasmid DNA should be purified using an anion-exchange column. 

 

1. Mix DNA in TE with  3.0M (NH4)2SO4, 100 mM Tris, 10 mM EDTA, pH7.0 to adjust to  '''2.1 M''' (NH4)2SO4. 

2. Load to column packed with PlasmidSelect Xtra resin, prequilibrated with '''2.0 M''' (NH4)2SO4, 100 mM Tris, and 10 mM EDTA, pH 7.5.

3. Wash column with 2.0M (NH4)2SO4, 100 mM Tris, and 10 mM EDTA, pH 7.5.

4. Elute supercoiled plasmid (SC pDNA) with '''1.7 M''' (NH4)2SO4, '''0.3 M NaCl,''' 100 mM Tris, 10 mM EDTA (pH7.5). 

5. Clean column with water. 

Endotoxin level should be less than 5 EU/mg. 

[[Category:Protocols]]

Plasmid MaxiPrep PlasmidSelect - Revision history

Paul.xu at 15:52, 16 October 2012