Patent Application
20080053911
The spin column of
In
In the alternative embodiment shown in
The following examples do not limit the scope of the invention. The effectiveness of the method according to the present invention is clearly demonstrated:
A suspension of insoluble polymers in AE buffer (0.5 mM EDTA; 10 mM Tris; pH 9.0) is set up and swollen over night at 4° C.
For the preparation of the spin columns the suspension was homogenized by vortexing and an aliquot was pipetted into the column. The gel bed was formed by a short centrifugation. This pipetting/centrifugation procedure was repeated until the height of the bed made about ¾ of the volume of the inner reservoir of the column.
Three spin columns were filled with different solid phase systems:
On each of the columns A, B and C 200 μL of a mixture of 100 μL water and 100 μL phenol/chloroform was placed. The columns were centrifuged for 30 seconds with 6000 g.
Only in the case of column A with no poly(vinylpolypyrrolidone) being present there was still a two-phase system left after the centrifugation step.
This result shows, that poly(vinylpolypyrrolidone) polymer is suited for adsorption of organic phase like phenol/chloroform and Sephadex G-10 does not lead to a separation.
5 μL pUC21-solution (5 μg pUC21), 10 μL 1M sodium chloride solution and 35 μL TE buffer (1 mM EDTA; 10 mM Tris; pH 7.5) were mixed with 50 μL phenol and 50 μL phenol/chloroform. The mixture was homogenized by vortexing and added to a spin column filled with adsorption medium, which was prepared by mixing the following components:
4 g Sephadex G-25; GE Healthcare; Cat No. 17-0032-01
1 g poly(vinylpolypyrrolidone)
1 g poly(ethylene-co-acrylic acid) sodium salt acrylic acid 5 wt.-%; Sigma-Aldrich Co. Cat No. 426733
in AE buffer (0.5 mM EDTA; 10 mM Tris; pH 9.0)
The column was centrifuged for 30 seconds with 6000 g.
A mixture of 25 μL of the column flow through, 4 μL 10×reaction buffer, 10 μL BiDest and 1 μL enzyme (1. BamHI; 2. HindIII; 3. Sau3A) was heated for 60 minutes at 37° C. in a water bath.
The results showed that after separation of the plasmid DNA the same could be linearized by restriction endonucleases and that hence a potential rest amount of phenol has got no influence on the enzymatic reaction of the restriction. In addition, it was shown that the salt was efficiently removed during the separation by gel filtration.
50 μL linearized pUC21-solution (Example 2) were mixed with 50 μL phenol/chloroform. The mixture was homogenized by vortexing and added to a spin column filled with adsorption medium, which was prepared by mixing the following components:
6 g Sephadex G-10
2 g poly(vinylpolypyrrolidone)
in AE buffer
The column was centrifuged for 30 seconds with 6000 g.
A mixture of 30 μL of the column flow through, 4 μL 10×reaction buffer, 5 μL BiDest and 1 μL T4 DNA ligase was kept for 150 minutes at room temperature.
The result showed, that after the separation the linearized plasmide DNA could be modified by ligation and a potential rest amount of phenol has got no influence on the enzymatic reaction of a ligation.
100 μL purified human gDNA (160 ng/μL) and 10 μL 5 M sodium chloride solution were mixed with 100 μL phenol/chloroform. The mixture was homogenized by vortexing and added to a spin column filled with adsorption medium, which was prepared by mixing the following components:
6 g Sephadex G-10
2 g poly(vinylpolypyrrolidone)
in AE buffer
The column was centrifuged for 30 seconds with 6000 g.
After the separation the gDNA could be amplified by PCR. This shows that a potential rest amount of phenol has got no influence on the enzymatic reaction of a PCR.
Classical phenol extraction: 100 μL sample+100 μL phenol/chloroform; vortexing (10 sec); centrifugation (30 sec); transferring supernatant in fresh tube; adding salt and ethanol; vortexing and centrifugation (10 min); discarding supernatant; adding 70% ethanol; centrifugation (3 min); discarding supernatant; drying pellet (10 min); resuspending in buffer.
Total duration: 25 minutes
Separation with Phase Lock Gel™: prespinning Phase Lock Gel™ tube (20-30 sec; 12000-16000×g); 100 μL sample+100 μL phenol/chloroform; mixing (vortexing not recommended) (10 sec); applying all to the column; centrifugation (5 min; 12000-16000×g); transferring (decanting) supernatant in fresh tube.
Total duration: 6 minutes
Separation according to the invention: 100 μL sample+100 μL phenol/chloroform; vortexing (10 sec); applying all to the column; centrifugation (30 sec; 6000×g).
Total duration: 1 minute
The duration of the separation process is clearly diminished when the method according to the present invention is used.
