Isolation of high molecular weight DNA using beads
Provided herein is a method for isolating high molecular weight (HMW) DNA using beads that are at least 200 μm in diameter that utilizes a device for retaining the beads and where the purified DNA eluant exits the device without shearing the HMW DNA. In some embodiments, the method comprises precipitating the DNA onto the beads, washing the beads in the device, and then eluting the DNA from the beads therein while substantially avoiding shear. Compositions and kits for practicing the method are also provided.
1. A method for purifying high molecular weight DNA comprising:
(a) obtaining:
(i) one or more centrifuge tubes for collecting fluid in a base from a bead retaining tube by centrifugation;
(ii) one or more bead retaining tubes that have a top opening oppositely placed to one or more exit openings, and a hinged lid that can close the top opening, wherein: i. the bead containing tube accommodates 1-25 beads of the beads per ml of volume of the bead retaining tube; ii, the one or more exit openings have a diameter that is smaller than the beads and allows DNA having a median length of at least 50Kb to pass through intact into the collection tube whereby the exit opening is not in contact with the base of the centrifugation tube; and
(iii) one or more beads that have a diameter of greater than 1 mm; and
(b) precipitating DNA from a DNA solution that comprises the high molecular weight DNA onto 1-25 beads in a first closed end tube;
(c) after removing liquid from the tube by aspiration and then washing the beads with a wash solution, transferring the beads into a first bead retaining tube;
(d) with the lid closed, centrifuging the bead retaining tube positioned in the first centrifugation tube to remove any residual wash solution;
(e) transferring the beads into a second closed end tube for adding eluant;
(f) transferring the eluant and the beads into a second bead retaining tube; and
(g) with the lid closed and the beads within, centrifuging the second bead retaining tube positioned in a second centrifuge tube, thereby collecting the purified high molecular weight DNA in the second centrifuge tube.
2. A method according to claim 1 , wherein the top opening of the bead retaining tube has a lip that engages with the entrance of the centrifugation tube.
3. The method of claim 1 , wherein the one or more beads having a shape that in one dimension has a diameter of greater than 1 mm up to 6 mm.
4. The method of claim 1 , wherein the high molecular weight (HMW) DNA in the eluant has a median length of at least 50 Kb.
5. The method of claim 1 , wherein the one or more beads are spherical glass beads.
6. The method according to claim 1 , wherein any of the first and second of the close ended tube is the same, the first and second of the bead retaining tube and the first and second of the collection tube are the same.
7. The method of claim 1 , wherein prior to (b) the method comprises agitating cells in a lysis buffer at 300-2000 rpm at a temperature in the range of 37° C.-56° C., wherein the speed of agitation determines the median length of the high molecular weight DNA precipitated on beads in (b).
8. The method of claim 1 , wherein (b) further comprises washing the high molecular weight DNA precipitated on beads for removing residual non-DNA material.
9. The method of claim 1 , wherein the bead retaining tubes have one or more exit openings having a diameter of less than the one or more beads, and the bead retaining tubes have designed so that the one or more beads do not block the one or more exit openings.
10. The method of claim 9 , wherein the one more exit openings have a diameter of at least 0.5 mm.