Vol II, Issue III Oct 2012 Multilogic in Science ISSN 2277-7601 An International Refreed & Indexed Quarterly Journal 11 www.ycjournal.net UNIVERSAL PROTOCOL FOR NUCLEIC ACID PURIFICATION FOR PLANT TAXA Kalyankar, V. B., Ughade, B.R., Khedkar*, G.D., Gupta, A. K., Tiknaik, A.D., Jamdade, R.A., Nalage, D. N. and Khedkar, T. S. Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Department of Zoology, Dr.BabasahebAmbedkarMarathwada University, Aurangabad, 431004 (Received : 09.08.2012; Revised : 20.08.2012; Accepted : 25.08.2012) Abstract Good quality DNA is the prerequisite for any type of biotechnological research. Several protocols are described by many researchers as well as quite a few dozen commercial DNA isolation kits are available in the market. Most of the protocols are very specific for typical taxonomic group and may not have similar quality of DNA with different taxa. Under this study we have developed a protocol which is universal for all plant taxa and can be used in isolation of DNA from live, dried and alcohol preservedtissues of variety of plants taxa.This protocol is relatively economical as it does not involve the RNAse-A and Proteinase-K use. It is environmental friendly as no use of phenol and non-hazardous since no use of liquid nitrogen. The protocol is proved to be less time consuming with better DNA yield from small tissue. Key words: DNA; plant taxa; tissue; CTAB; PVP; rbcL; sequence. Introduction In molecular biology or biotechnological research, obtaining good quality DNA is the prerequisite and important step (Kumari, et al. 2012).Recently, vascular plant systematics has begun to include studies of DNA variation as one of the common methods of analysis (Chase and Hills, 1991).Obtaining DNA from plant resources is cumbersome and becomes complicated due to presence of many active principles. It includes the polysaccharides, polyphenolic compounds and other secondary metabolites whichcoprecipitate with DNA in the extraction procedure and inhibit DNA digestion and PCR (Zhange andMcStewart, 2000). The renowned use of CTAB for purification of DNA from plants is historic but is applicable specifically in angiosperms(Murray and Thompson, 1980).The present protocol is the developed by modifying the CTAB protocol (Murray and Thompson, 1980) and tested fordifferent plant taxa and prove to be better in DNA quality, quantity, quicker as well as cost effective as compared to options available to date. Material methods Plant material collectionThe fresh plant material was collected and preserved in 95% ethanol (Pyle and Adams 1989)during June, 2011 to October, 2012. It includes, dry leaves, bark, stem and hard wood. The representative plants used for the present study were Spirulinarobusta (Algae), Ricciahimalensis (Bryophyte), Nephrolepisexaltata (Pteridophyte), Thujaorientalis (Gymnosperm), Bambusavulgaris(Monocot) andAcalyphahispida (Dicot). Reagents and chemicals Extraction buffer:1M Tris-HCl5ml (pH 8.0);0.5M EDTA, 2ml (pH 8.0);5M NaCl, 17.5ml;10% CTAB (Cetyl-trimethly-ammonium-bromide)10ml, (5gm in 50ml);double distilled water, 15.5ml to make the volume 50ml and adjust the pH to 7.5to 8.0. Autoclave the whole content before use. PolyVinylPyrrolidone (PVP)(molecular weight 30- 40kd) 20g/l of CTAB buffer added just before use. mercapto ethanol (2 mercapto ethanol). 20% SDS (Sodium-dodacyl-sulphate)v/v in water. 24:1 Chloroform: Isoamyl alcohol made by mixing the two solutions together v/v. TE buffer: 10mM Tris and 1mM EDTA mixed at pH: 8.4. Protocol details Take small quantity of plant tissue (approximately 100mg, 0.5-1cm 2 ) and dry it on clean tissue paper, while dealing with angiosperms, mid rib region of the leaves must be removed; transfer it to microcentrifuge tubes (1.5 ml).Add to it 600μl of pre-warmed (60 0 C) CTAB buffer (extraction