Isolation of DNA from Plant TissueAim To isolate DNA from plant tissue and to determine its purity.

Principle The cell wall must be broken or digested to release the cellular components. This is doneby grinding the tissue in liquid nitrogen with a pestle and mortar. Usually phenolextraction yields a fairly pure DNA sample. This treatment however may not be sufficientto give pure DNA if the cells also contain significant quantities of other biochemicals likethe plant tissue which often contain large amounts of carbohydrates that are removed byphenol extraction. DNA from the plant tissue is therefore usually obtained by using adetergent either SDS or CTAB. CTAB forms insoluble complex with nucleic acids(Nucleic acid CTAB complex) leaving behind carbohydrates, protein and othercontaminants in the supernatant. The insoluble nucleic acid-CTAB complex precipitate isthen collected by centrifugation and re-suspended. EDTA in the lysis buffer inactivatesendogenous nucleases by chelating Mg 2+ ions. The tissue buffer mixture is emulsifiedwith chloroform phenol to denature and separate remaining proteins from the DNA.The genomic DNA can be precipitated by using absolute ethyl alcohol. Materials Microfuge tubes, centrifuge, oven, EDTA, Tris, CTAB or SDS, Chloroform, Ethanol,isoamyl alcohol, isopropanol, sodium acetate buffer. Methodology Around 0.5 g of fresh leaf tissue or cauliflower was ground into powder in a precooledpestle and mortar.Exactly 9 ml of pre-heated CTAB extraction buffer was poured on to the leaf powder in apolypropylene centrifuge tube. The mixture was gently inserted several times andincubated for 60 minutes in an oven at 65 0 C. the tubes were gently agitated during theincubation time at regular intervals.The tubes were cooled after incubation period and 5 ml of chloroform

 isoamyl alcohol(24:1) was added and mixed gently for 5-10 mts.The tubes were spun at 3000rpm for 10 minutes.The layers were clearly seen after centrifugation. The top aqueous phase was poured off into new tube and re-extracted with chloroform-isoamyl alcohol.To the aqueous phase 6 ml of cold isopropanol was added and inverted gently toprecipitate the nucleic acid. The precipitate was dissolved with 1 ml of TE buffer and re-extracted using phenol chloroform (1:1) at 3000rpm for 10 minutes. The aqueous phase was transferred to a newtube.About 1 ml of chloroform: isoamyl alcohol was added to the aqueous phase and spun at3000rpm to separate the phase.To the aqueous phase 50 ml of 3M sodium acetate and 2.5 ml of absolute ethanol wasadded to precipitate the DNA.The tube was gently inverted for several times and incubated at 20 0 C for 20 minutes.The pellet in the microfuge after centrifugation was collected with 70% ethanol. Ethanolwas discarded and the pellet was air-dried. Dry powdered form of DNA was collected aspellet was then dissolved with 150 l of TE buffer. Estimation of the concentration of DNA DNA has a maximum absorbance at 260nm. Based on the extinction co-efficient, anoptical density of 1.0 at 260 nm corresponds to 50g/ml of double stranded DNA. Theratio of O.D 260/280 provides the estimate of purity. A typically pure DNA has a rationof approximately 1.8. Ratio less than 1.8 indicates the probable presence of proteins orother UV absorbers. Ratio higher than 2.0 indicates that the sample may be contaminatedwith chloroform or phenol and should be reprecipitated with ethanol. Methodology To measure the concentration of DNA, the sample was diluted with TE buffer. The spectrophotometer was blanked using TE buffer. Absorbance of the sample at 260nm was recorded and the DNA concentration wascalculated from the following relation.Conc. of double stranded DNA = O.D (260) X 50 (g/ml) X Dilution factor / 1 O.D (260) Result: White threads of DNA was precipitated and stored in TE buffer. The DNA was thensubjected to agarose gel electrophoresis and DNA band was visualized using ethidiumbromide. Discussion

Organic extraction is a conventional technique that uses organic solvents to extractcontaminants from cell lysates. The cells are lysed using a detergent, and then mixed withphenol, chloroform, and isoamyl alcohol. The correct salt concentration and pH must be used during extraction to ensure that contaminants are separated into the organic phaseand that DNA remains in the aqueous phase. DNA is usually recovered from the aqueousphase by alcohol precipitation. This is a time-consuming and cumbersome technique.Furthermore, the Methodology uses toxic compounds and may not give reproducibleyields. DNA isolated using this method may contain residual phenol and/or chloroform,which can inhibit enzyme reactions in downstream applications, and therefore may not besufficiently pure for sensitive downstream applications such as PCR