Study of Hygienic practices of street food vendors in ... - Shiats.edu.in

Aradhana Irene Charan, Nishant Kumar Srivastava, Sanjay Singh, Amit Alexander Charanand Abhishek Sharanacids, making it a richer source of proteins compared to other vegetables. The drumsticktree in fact has the highest protein ratio of any plant on earth, with calcium content of theleaves being 297 mg per 100 g of leaves. 100 g of freshly cooked drumstick leavesprovide for 75 % of iron and 50 % of proteins required by a growing child.MATERIALS AND METHODFresh leaves of M. oleifera were collected from Germplasm Bank established atNational Bureau of Plant Genetic Resources (NBPGR), Ranchi, which contains accessionof drumstick plants which were collected from different locations of Jharkhand. Extractionof DNA in M. oleifera was done by (Doyle and Doyle, 1990), with some modificationin the procedure, as M. oleifera contains some phenolic compounds which causestickiness in the sample and make it difficult to isolate the DNA . So, in context withabove an additional step is added, which uses wash buffer containing 0.1% PVP and4% - Mercaptoethanol to wash out the phenolic compounds (Hedrick, 1992).The genomic DNA was then subjected to spectrophotometric quantification at 260nm and 280 nm. 14 eppendorf tubes were taken each having 1 ml volume consisting of980 µl TE buffer and 20 µl DNA sample and another 1 ml eppendorf tube which is usedas blank consisting of 1000 µl TE buffer. Agarose gel electrophoresis was also done tocheck the presence of DNA in the sample. RAPD analysis was done with total sevenprimers which were already available in the laboratory and they were selected on thebasis of their genetic diversity and identity (Table 1). PCR was performed with followingsettings- total 18 µl in which 1.5 µl dNTPs, 0.5 µl Taq Polymerase, 1.0 µl Taq Buffer, 1.5µl Primer, 0.5 µl MgCl 2, 11 µl PCR grade water and 2 µl of DNA sample. Total 45 cycleswere performed. Amplification products were analyzed by electrophoresis using 1.5%Agarose gel in 1X TAE running buffer. The bands were visualized under the GelDocumentation System. Bands were scored, and analyzed with software POPGENEVersion 1.32 (32-bit).RESULTS AND DISCUSSIONIn preliminary screening, seven primers were chosen for the evaluation of geneticdiversity among the M. oleifera populations. Specific DNA band patterns were observedwith all the seven primers used. A total of twenty five bands were scored and all werepolymorphic (Arnold and Emms, 1998). On average each primer produced 3.2 RAPDbands, the largest number was obtained from RPI 8 and RPI 15 and lowest number wasobserved from RPI 5 after running on Agarose gel. UPGMA cluster was prepared usingNei's Genetic distance method UPGMA (Nei, 1973) which is modified from NEIGHBOR107