isolasi dna kromosom_3
TRANSCRIPT
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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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PRAKTIKUM BIOLOGI SEL, 2011 5
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.
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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
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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