Filtration of DNA and Bugs @ INT
Two experiments were performed to explore application of SiMPore filters within INT’s microfluidic cartridges to allow the capability for pathogen type specificity in nucleic acid purification.
1) The first experiment tested the efficiency at which nucleic acids could pass through the membranes. T4 polynucleotide kinase was used to 32-P radiolabel two different sized PCR products: 200 basepair (bp) and 6800 bp DNA. About 50,000 cpm of radiolabeled DNA was used to spike 10 micrograms of unlabeled PCR product of the same size; final solution volumes were 500 ml. SiMPore SepCon-40 (40 nm pore) spin columns were filled with the entire solution prepared for each 32-P spiked DNA sample. The columns were then spun at 2000 rpm for 2 minutes. Under these centrifugation conditions, approximate 30 to 50 ml of solution passed through the column and was saved for analysis
To determine how well each DNA size passed through the SiMpore filters, 10 ml aliquots from the column filtrate and from the solution that still remained unfiltered in the column were taken, and then suspended into 6 ml of EcoScint Scintillation fluid for counting in a Beckman Scintillation Counter. The measured counts per minute (cpm) per 10 ml of add filtrate or retentate was essentially the same ( + 2%), which indicates that both 200 and 6800 bp size DNA were not restrict while being filtered by 40 nm SiMPore microchips.
2) In the second experiment, the ability of SiMPore SepCon-40 and SepCon 550-600nm columns to retain either E. coli cells or B. subtilus spores was tested. Serial dilutions of these bacteria were prepared from freshly cultured E. coli cells or refrigerated stocks of B. subtilus spores to concentrations of 10^6, 10^5, and 10^4 cells per ml. Fifty ml of each dilution and bacterial source were suspended into 450 ml of 50 mM phosphate buffer (pH 7.8) and centrifuged at 2000 rpm for 2 to 3 minutes in SepCon columns of both pore sizes. Nutrient agar plates were plated with 10 ml of column filtrates or with 10 ml of a ten-fold dilution of the filtrate (to facilitate the counting of colonies). Plates were also prepared with straight and diluted aliquots of the E. coli and B. subtilus spores from serial dilutions that were made in parallel for controls (not spun through SepCon columns), to obtain counts of colonies expected for 100% cell or spore flow through.
Both B. subtilis spores and E. coli cells are larger than pores of the SiMPore –SepCon filters tested; the observation of no colony growth on nutrient plates for all cell and spore concentrations passed through SepCon columns demonstrated complete effectiveness in retention of both forms of bacteria. The control plates had colony counts within the range routinely expected for the dilution applied and the calculated cell concentration of the starting E. coli culture and B.s. spore stocks; these controls validated the viability of the cells and spores used in the experiment.