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Purification of labeled DNA
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After labeling and/or DNase treatment, the labeled DNA can be purified by passage through a Sephadex spin column, by ethanol precipitation, phenol extraction or using Microcon spin columns (also discussed here). These procedures remove unincorporated nucleotides, enzymes and salt. FISH using cosmids, BACs or YACs can be performed without prior probe DNA purification. For example, after nick translation, 2-3 µl reaction (usually 20-40 ng/ul cosmid, BAC DNA) can be directly used for FISH, by mixing them with 8-9 µl hybridization buffer and 1-2 µl concentrated (10 µg/µl) human Cot1 DNA. Hybridization results were identical and background was the same, regardless whether the labeled probe was first purified. Exceptions to this observation were TexasRed, Cy3.5 and Cy5.5 nucleotides (or free dyes) which appear to bind non-specifically to the biologic material on the slide, probably because of their charged molecule.
Note: when using large amount of labeled probe DNA, such as for M-FISH or CCK, cleaning up the DNA by phenol extraction or another procedure does decrease background and improves hybridization results.
Purification of labeled DNA is particularly important for removing the free dye, when fluor-labeled nucleotides are custom-prepared in the laboratory (for details on nucleotide labeling click here).
Sephadex G-50 spin columns
Spin columns can be custom made in the laboratory. 10-20g sephadex G-25 or G-50 powder (Pharmacia Biotech) is resuspended in 300-500 ml of a common buffer (for example PBS or TE, pH7-7.5) or distilled water and is sterilized by autoclaving. The columns can be made in 1cc (for 20-50 ul probe volume) or 3cc (for up to 150 ul) disposable syringes (Beckton Dickinson). After the piston is removed, the narrow end of the syringe is plugged by dropping one glass bead (2mm diameter, Sigma) inside. The use of a glass bead is much easier and more convenient than glass wool. The syringe is placed vertically inside a 15 ml tube, and is filled with buffer (PBS, TE) followed quickly by Sephadex suspension. This sequence of steps prevents air bubble formation in the column. Sephadex is added and accumulates until it fills the syringe to about 0.5 cm from the top. The 15 ml tube holding the column is placed in a centrifuge and spun 20-30 seconds each at 400 rpm and 1000 rpm and 2 minutes at 2000 rpm (300x-400x g). This stepwise increase in centrifugation speed is necessary, as the Sephadex will be gradually sedimented and will not leave the column (a sudden burst of 2000 rpm may result in some of the Sephadex leaking out of the column). A 1.5 ml collection vial with no lid is dropped inside the 15 ml tube, and the column tip is inserted so that its end rests inside the vial. The DNA probe is pipetted on top of the column, which is then subjected to the same centrifugation steps. The purified DNA probe accumulates in the vial, while the labeled nucleotides remain in the column. No significant losses of labeled probe DNA are recorded with this procedure.
Phenol extraction
If proteins or enzymes are present in the labeled DNA probe solution, especially after nick translation, phenol extraction can be used. Phenol extraction is performed by mixing the probe (1 volume) with 1/2 volume phenol and 1/2 volume chlorophorm:isoamylalcohol 24:1, followed by vortexing and centrifugation, 2 minutes at maximum speed on a tabletop microfuge (13000-14000 rpm). DNA labeled with derivatives of rhodamime or with DIG should not be subjected to phenol extraction, as there are significant losses of labeled probe in the phenol phase (also discussed here).
Partial purification by ethanol precipitation
An easy procedure for separating the DNA probe from the free nucleotides is the common ethanol (2.5 volumes) or isopropanol (0.8-1 volume) precipitation. This allow concentrating the DNA, while separating most of the free labeled nucleotides and salt which are removed in the 65-70% ethanol solution.
A convenient procedure used often in our laboratory, is to precipitate the DNA in 1.5-2 volumes of a 1:1 mixture of ethanol:isopropanol. No added salt is needed with nick translation or PCR reactions. Precipitation by ethanol, isopropanol or their mixture requires only about 10 minutes at room temperature, and is followed by 10 minute spin in a tabletop centrifuge, at 14000 rpm to pellet the DNA. Precipitation at -20 C or -80 C did NOT improve DNA recovery.
After inverting the tube and draining the ethanol out, the DNA pellet can be conveniently dried in a heat block at 50-60 C, for 5-10 minutes or until the ethanol evaporates.
Microcon columns (Millipore Corp)
We have successfully used Microcon YM-30 or YM-50 to remove unincorporated nucleotides or salt from labeling reactions. However, proteins are not removed by these columns, as they have a high molecular weight (see manufacturer indications).