isolasi dna kromosom_3

7/31/2019 Isolasi Dna Kromosom_3

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1PRAKTIKUM BIOLOGI SEL, 2011


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Tujuan

Mahasiswa dapat melakukan isolasi DNAkromosom tanaman

Mahasiswa dapat melakukan analisiskuantitatif hasil isolasi DNA kromosomtanaman menggunakan metodespektrofotometri

PRAKTIKUM BIOLOGI SEL, 2011


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PRAKTIKUM BIOLOGI SEL, 2011 3

Physical Characteristics of DNA

DNA absorbs UV light at 260 nm Allows quantitation

DNA is water soluble

DNA precipitates in alcohols DNA carries a net negative charge


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DNA Kromosom


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Resuspend & lyse cell walls. Ice cold TES (Tris, EDTA,

NaCl).

EDTA is a chelating agent and has great affinitywith matel ions and Mg-ion present in DNase asa cofactor and responsible for DNase action thatdegrade the DNA,here EDTA bind with Mg-ionand nullyfy the action of Dnase

Tris is the main buffering component. Trisinteracts with the LPS (lipopolysaccharide) in the

membrane, serving to destabilize the membrane

lysozyme (degrades peptidoglycancell walls)

Proteinase K (to degrade protein

that are bound to the DNA molecule) Enzymes (RNAses ) are often

added to degrade the RNA in thesample for purification

SDS, CTAB(detergent to lyse thecells)

Structure of a Gram-Negative Cell Wall

The DNA in the nucleus is surrounded by proteins


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CTAB

Cetyltrimethylammonium Bromide

cationic detergent.

CTAB has been used for the isolation of plant high

molecular weight DNA.

8PRAKTIKUM BIOLOGI SEL, 2011


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The cell and nuclear membranes have beenbroken apart,

as well as all of the organelle membranes,such as those around the mitochondria and

chloroplasts.

So what is left?

Proteins Carbohydrates (sugars)

lipid DNA


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Problems encountered in the isolation andpurification of high molecular weight DNA from

plant species

(1) degradation of DNA due to endonucleases

(2) inhibitor compounds like polyphenols and othersecondary metabolites which directly or indirectlyinterfere with the enzymatic reactions (Weishing et al.,1995).

The presence of polyphenols, which are powerfuloxidising agents present in many plant species, can

reduce the yield and purity of extracted DNA (Loomis,1974; Porebski et al., 1997).



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B-mercaptoethanol

a strong reducing agent whichcan remove tannins and otherpolyphenols often present in thecrude plant extract


2-Mercaptoethanol reacts with aldehydesand ketones to give the correspondingoxathiolanes
http://en.wikipedia.org/wiki/Aldehydehttp://en.wikipedia.org/wiki/Ketonehttp://en.wikipedia.org/wiki/Ketonehttp://en.wikipedia.org/wiki/Aldehyde


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B-merkaptoethanol :Cleave disulfide bond of protein



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Extract and purify DNA

Separation of DNA from other molecules

Chloroform extraction


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Sentrifugasi

DNA & RNADilarutkan

TE


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Extract and purify DNA

Alcohol precipitation of nucleic acids

Need to have at least 0.2M of monovalentcation to the shield negative charges onDNA (sodium acetate pH 5.2)

Ethanol.

Precipitation temperature normally at 0

o

Cor room temperature


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Hook or Spool out

Chromosomal DNA


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Khanuja et al, 1999

isolasi dna kromosom_3

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