Methods and devices for nucleic acid purification
The invention provides pipette tip columns and automated methods for the purification of nucleic acids including plasmids. Nucleic acids can be purified from unclarified, clarified or partially-clarified cell lysates that contain cell debris. The columns typically include a bed of medium positioned in the pipette tip column, above a bottom frit and with an optional top frit. Plasmid preparation scales include miniprep, midiprep, maxiprep, megaprep and gigaprep.
1. A method for capturing plasmid DNA on a column, comprised of:
a) providing the column, wherein the column is comprised of a solid phase;
b) providing an unclarified cell lysate comprised of a liquid and a flocculent, wherein the liquid contains plasmid DNA;
c) providing a large-pore filter having a pore size, wherein the pore size of the large-pore filter is greater than 50 μm;
d) partially removing the flocculent by passing the unclarified cell lysate through the large-pore filter using gravity flow to produce a partially clarified lysate, the partially clarified lysate including remainder flocculent that passes through the large-pore filter; and
e) passing the partially clarified lysate over the column, wherein a portion of the plasmid DNA is captured on the solid phase and the remainder flocculent at least partially flows freely through the column.
2. The method of claim 1 , wherein the pore size of the large-pore filter is greater than 200 μm.
3. The method of claim 1 , wherein the partially clarified lysate is comprised of between 2% and 40% (vol/vol) of the remainder flocculent.
4. The method of claim 1 , wherein the solid phase is comprised of silica or an anion exchange resin.
5. The method of claim 1 , wherein the unclarified cell lysate is comprised of a chaotropic solution.
6. The method of claim 1 , wherein step (e) is automated.
7. The method of claim 6 , wherein step (d) is automated.
8. The method of claim 7 , wherein the large-pore filter is designed to filter a portion of particulates included in the unclarified cell lysate using gravity flow and wherein step (d) is performed in 15 minutes or less.
9. The method of claim 8 , wherein step (d) is performed in 10 minutes or less.
10. The method of claim 8 , wherein the method is performed in parallel on between 2 and 24 samples.
11. The method of claim 1 , wherein the following steps are performed after step (e):
f) passing a wash buffer through the column, and
g) eluting the plasmid DNA by passing an elution buffer through the column, wherein the eluted plasmid DNA is substantially endotoxin-free.
12. The method of claim 11 , wherein the eluted plasmid DNA has an endotoxin concentration of less than 50 endotoxin units per microgram.
13. A method for capturing plasmid DNA on a column, comprised of:
a) providing the column, wherein the column is comprised of a solid phase;
b) providing an unclarified cell lysate comprised of a liquid and a flocculent, wherein the liquid contains plasmid DNA;
c) providing a large-pore filter having a pore size;
d) partially removing the flocculent by passing the unclarified cell lysate through the large-pore filter using gravity flow to produce a partially clarified lysate, wherein the partially clarified lysate is comprised of between 3% and 40% (vol/vol) of remainder flocculent that passes through the large-pore filter; and
e) passing the partially clarified lysate over the column, wherein a portion of the plasmid DNA is captured on the solid phase and the remainder flocculent at least partially flows freely through the column.
14. The method of claim 13 , wherein the pore size of the large-pore filter is greater than 200 μm.
15. The method of claim 13 , wherein the solid phase is comprised of silica or an anion exchange resin.
16. The method of claim 13 , wherein step (e) is automated.
17. The method of claim 16 , wherein the large-pore filter is designed to filter a portion of particulates included in the unclarified cell lysate using gravity flow and wherein step (d) is performed in 15 minutes or less.
18. The method of claim 17 , wherein the method is performed in parallel on between 2 and 24 samples.
19. The method of claim 13 , wherein the following steps are performed after step (e):
f) passing a wash buffer through the column, and
g) eluting the plasmid DNA by passing an elution buffer through the column, wherein the eluted plasmid DNA is a substantially endotoxin-free.
20. The method of claim 19 , wherein the eluted plasmid DNA has an endotoxin concentration of less than 50 endotoxin units per microgram.