ISOLATION OF GENOMIC DNA FROM PLANT A seminar submitted at

MITS School of Biotechnology

Affiliated Under

UTKAL UNIVERSITY, BHUBANESWAR

is given by

GOPAL KRUSHNA SOREN

Roll no: 1302010190490015

from B.Sc. 3rd yr. 2013-16 batch.Email id:- gopalkrushnasoren1815@gmail.comContact no.-9437395781.

Contents:-1. Introduction2. Safety guidelines3. Experimental outline4. Materials5. Pre-lab preparation6. Method

I. Day 1II. Day 2

7. Application of this technique8. Conclusion9. Bibliography10.Declaration

Introduction::The Objectives of this of module are:1. To isolate genomic DNA from shoot tissue of Pea(Pisum sativum).2. Compare it to DNA isolated from chloroplasts of Pea.

Isolation of DNA from plant tissues is at the heart of plant molecular biology. Because plant cells are surrounded by rigid cells walls & also plant tissues often contain a variety ofsecondary metabolites that can damage DNA.

DNA isolated by methods such as the one presented here represents total cellular DNA.In plants, this means the isolation of three distinct genomes:I. The nuclear genomeII. The chloroplast genomeIII. The mitochondrial genome

Generally, when we talk about plant DNA we mean nuclear DNA, but it is important to remember that chloroplasts & mitochondria each have distinct genomes.

Materials:-EB(extraction buffer): 50mM Tris acetate pH 8.0, 1% CTAB(Cetyltrimethyl ammonium

bromide), 50mM EDTA, 1mM 1,10-O-phenanthroline, 0.7 M NaCl, 1% betamercaptoethanol.

Chloroform.

Isopropyl alcohol.

80% ethanol, 15mM ammonium acetate, pH 7.5

TE(Tris-EDTA) buffer: 10mM Tris pH 8.0, 1mM EDTA

Centrifuge tubes(capped, 50 ml capacity)

Water bath 65°C.

Safety Guidelines:-

Reagents used in this protocol are potentially dangerous.

Cetyltrimethyl ammonium bromide (CTAB) in the extraction buffer

is a strong detergent and can cause burns to the skin. Chloroform

is toxic by inhalation or on contact with skin. Follow your

Instructor’s direction for proper handling and disposal of these

materials.

Experimental OutlineDay 1. Grind tissue & suspend in extraction

buffer (EB). Incubate at 65°C for 1 hr. Extract with chloroform. Precipitate DNA with isopropyl alcohol. Re-suspend the DNA in buffer & store.

Day 2.Compare this DNA

preparation with DNA isolated from chloroplasts by gel electrophoresis.

IUPAC name: hexadecyl-trimethyl-ammonium bromide

Chemical formula: C19H42BrN or {(C16H33)N(CH3)3Br}Appearance: white powder

Melting point: 237-243˚C(510-516K)

Molar mass: 364.45 g/ml

Cetyl-Trimethyl-Ammonium Bromide (CTAB) is an amine based cationic quaternary surfactant.The centrimonium or hexadecyltrimethylammonium cation is an effective antiseptic agent against bacteria and fungi. Its uses also include providing a buffer solution for the extraction of DNA.

Importance & Properties of CTAB

Structure of CTAB

Fig showing the distinct DNA band on Electrophoresis after using CTAB method in w.r.t. others

Day 1:- Grind 1 gm. of freeze dried tissue to a fine powder with mortar & pestle. Fine powder + 25 ml of EB in 50 ml capped centrifuge tube. Put tube in 65°C water bath & incubate there for 1 hr. Take out the test tube & cool it. Centrifuge the tube at greater speed of 3500×g for 10 min. Add 20 ml of chloroform to EB of the tube & mix thoroughly. As given in the fig.1 distinct layer was formed. Green layer of chloroform contain bits of plant

pellet. Intermediate Y+G layer contains denatured protein. At the straw yellow region of the tube majority of DNA is present. That will taken by micropipette avoiding the interphase material of centrifuge tube.

To the aqueous phase add 2/3 vol. of isopropyl alcohol. DNA will precipitate to form a cottony mass.

Transfer the DNA to a clean flask. Add 10 ml of 80% ethanol, 15mM ammonium acetate and swirl to wash.

After 20min., transfer the precipitated DNA to a micro centrifuge tube and centrifuge briefly to drive the DNA to the bottom of the tube.

P.T.O

Methods

continue Remove the residual Ethanol using a micropipette. Allow the DNA in uncapped tube for about

10min. on the table. Add 75ml. Of TE buffer to the DNA for dissolving the precipitate.

DNA

Fig. 1 Fig. 2Schematic diagram for Day 1

DAY 2:-Separate 2µl of DNA from the stock solution.Cut that DNA with a restriction endonuclease enzyme.On an Agarose Gel, compare total DNA (both cut & uncut) with DNA isolated from chloroplasts (both cut & uncut).

Precautions:-

1. At the end of Day-1; leave the capped tube of DNA in the refrigerator so that in Day-2 large quantities of DNA will dissolve completely.

2. If uses of chloroform is neglected then Grind tissue as before, mix with EB and incubate in the water bath.

3. Centrifuge to pellet undigested plant tissue. Transfer the supernatant to a fresh tube/flask/beaker and proceed as according to the protocol.

APPLICATION OF THIS TECHNIQUE:

For determination of Evolutionary Relationship of a Plant with it’s Ancestors.

Study of origin of certain plants species. Used in Molecular biology. Used in Recombinant DNA technology. Hybridization experiment of DNA.

And so many application regarding with Plant’s genomic DNA.

Conclusion:

Observing the Agarose gel in the presence of UV light results the visualization of bright red-orange band of due to the fluorescence of Bromo Phenol Blue and Ethidium bromide.

Cut DNA are present far away from the starting point & uncut DNA are ran small distance in gel due to large size.

This experiment would be a compulsory experiment in biology background stream so that student can perform good in laboratory accepts.

This experiment should well known by all student because it would help them in future.

It could be a common experiment in Plant biotech & Molecular biology subjects.

BIBLIOGRAPHY https://

www.google.co.in/search/plant+genomic+dna+extraction https://

www.google.co.in/search/CTAB+method+of+plant+genomic+dna+extraction

https://www.google.co.in/structure+of+CTAB Laboratory manual on biotechnology(P.M. Swamy) 2008-09 1st

edition Concept of Botany (XI & XII){Bendre & Pandey} 4th edition 2012

THANK YOUDeclaration- I do hereby declare that this seminar report generate by me with the help of above regarding URLs and references. I will take the responsible for any kind of mistakes on above. I had delivered my seminar on 23 October 2015. I am submitting my seminar report on 16 March 2016 at MITS school of biotechnology.

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