Recently, our lab began using alpha-P32 dCTP-labeled oligonucleotides to screen a BAC library (actually a high-density nylon membrane that we had purchased from Resgen). To purify the radio-labeled probes, we began using GE Healthcare’ NAP-25 columns. These columns are pre-packed, disposable columns designed for the rapid and efficient desalting, buffer exchange and purification of DNA and oligonucleotides (equal or greater than 10 mers) utilizing gravity flow (as opposed to the spin-column format). The whole process of purifying DNA takes less than 30 minutes, depending on how many columns are being used at the same time. The columns are pre-packed with Sephadex G-25 DNA Grade in distilled water (which contains 0.15% Kathon CG/ICP Biocide, used as a preservative) and can be stored at room temperature. They come in different sizes for purifying different volumes of samples: 0.5 ml (NAP-5), 1 ml (NAP-10), and 2.5 ml (NAP-25). This review is specifically for the NAP-25 Columns, which were recommended for purifying radioactive labeled DNA for use in probing a cDNA library membrane.
The NAP-25 Column kit comes with 20 individual, one-time use columns. The gel bed dimensions are 1.5 x 4.9 cm, the maximum sample volume is 2.5 ml, and the volume of eluted sample is about 3.5 ml. The yield and purity as stated by GE Healthcare should be greater than 90% with less than 3% salt contamination. The use of the NAP-25 columns are very easy requiring little time and only one additional reagent, the equilibration buffer, 10 mM sodium phosphate buffer, pH 6.8 (that must be prepared to use the columns). The recipe for the equilibration buffer can be found in Current Protocols of Molecular Biology or other molecular biology protocol books.
Purification of DNA using NAP-25 is very easy and quick. The columns must be equilibrated with 25 ml, which generally takes about 10 minutes as the columns can only take about 5 ml of fluid at a time. Once the column has been equilibrated, the probe is added in 2.5 ml of fluid. An additional 3.5 ml of equilibration buffer is then added and 1.5 ml fractions are collected. The samples with the most incorporated label are then identified, pooled and used for probing the membranes. The columns themselves are simply discarded.
Purifying probes using the GE Healthcare’ NAP-25 column is simple, efficient and a relatively safe way to purify radiolabeled oligonucleotides. Overall, we are pleased with the way these columns work and would not have a problem recommending them.
Hee Chul Lee
Graduate Student
Dept. of Biochemistry
NYU School of Medicine