In this assay plasmid DNA was exposed to indoor PM samples and DNA strand breakage was quantified by gel electrophoresis technique. In this assay a specific hydroxyl radical scavenger i.e. Mannitol (4 mM) was used to protect the DNA scission and measure the role of the hydroxyl radical in the degradation of the super coiled DNA (Murrant, 2001). In this assay ¼ of filter was cut off and immersed in 1 mL phosphate-buffered saline (PBS). The samples were first ultrasonicated for 15 minutes followed by shaken gently and then vortexed for 15 minutes. This released the particulate matter (PM) from the filter substrate into the solution. PM fractions were taken into an EP (eppendorf) tube and each sample was dilutes with PBS to the required concentration. Extracts of PM was tested for their ability to damage DNA in supercoilled phage DNA. 2.3.3.1.1 Standard Procedure for Plasmid DNA assay Gel electrophoresis of plasmid DNA is a method used to separate DNA in a matrix of agarose and this technique involves the following steps (i) Preparation of Gel In this study 8% agarose gel was prepared by dissolving 0.642gm agarose in 40 ml 1X TBE (tris borate EDTA) buffer. …show more content…
The major sources of error concerning the sampling and analyses of particulate matter samples include (i) artifacts or contamination of samples, (ii) loss of collected particulate matter species during sampling or after sampling, (iii) sample handling, transport and storage, (iv) modification of samples during analyses, and (v) errors in data handling. In order to control and minimize the overall uncertainty caused by these factors, the sampling of PM and weighing of filters were carried out according to a standard operation procedure to assure high quality of sample