DNA molecules are often part of solutions or lysates that require clarification by filtration. To test whether filtration membranes bind dilute DNA samples, radioactively-labeled PCR products were added to Tris EDTA (10 mM Tris, 0.1 mM EDTA, pH 7.0) buffer. Samples were filtered using the same general handling procedures discussed previously for the analysis of protein binding. Biodyne B membrane is a transfer membrane designed to bind DNA, making it a good choice for a positive control for binding during filtration.
Double-labeled PCR products were synthesized from primers corresponding to a 400 bp fragment to pUC18. Free nucleotides and primers were removed from the pooled PCR products by using a 100K Nanosep® device to concentrate the DNA. The amount of radiolabeled DNA present was determined by gel electrophoresis, and specific activity was determined by counting to be approximately 540 CPM/ng. A 500 ng sample of purified-labeled DNA was diluted to 5.0 mL in TE buffer (10 mM Tris pH 7.0, 1 mM EDTA). A sample containing 5.0 mL of the 100 ng/mL DNA solution was filtered, air-purged and rinsed with 1.0 mL TE and counted in a multilabel counter.
As with the protein binding data, the transfer membrane controls bound the majority of the test sample even though the exposure during filtration was brief. Similar results were seen with other binding membranes (not shown). Supor and GHP membranes bound very low quantities of DNA, similar to that seen for the competitor’s low-binding membranes. Filtration of dilute DNA solutions with most low binding microfiltration membranes should not cause significant DNA sample loss.