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 DNA Isolation from plant sample by CTAB

method
 Causes of using chemical compound in DNA
isolation

• What is “TE”stands for?


Tris-EDTA (Ethylenediamine Tetraacetic Acid; buffered solution).
"TE" is derived from its components: Tris, a common pH buffer, and
EDTA, a molecule chelating cations like Mg2+. The purpose of TE buffer
is to protect DNA or RNA from degradation.

• What is the role of TE buffer in DNA isolation?


The purpose of TE buffer is to protect DNA or RNA from degradation. It
is a buffer for storage of DNA & RNA.

Role of phenol,chloroform in DNA isolation?


Phenol- phenol is used to denature the proteins. Or
phenol helps to remove non polar proteins and lipids from the solution.
chloroform - prevent shearing of dna during isolation. It solubilizes
lipids and a lot of proteins to remove them from the DNA.

Phenol -chloroform and isoamylalcohol role in dna


isolation?
The phenol, chloroform and also isoamyl alchohol are added in a specific ratio
of 25: 24:1.
Isoamylalchohol, (CH3)2CHCH2CH2OH or Isopentanol :Used to prevent
phosgene(COCl2, Carbonyl chloride) from reaction of chloroform(CHCl3) with
air.

The Phenol:Chloroform:Isoamylalchohol (PCI) solution is added to the cell


extract after removal removal of debris. After proper mixing, cetrifugation is
done to separate the phases. Two phases are formed: The upper, the
aqueous phase that contains DNA, the lower phase, that phenol phase, that
contains organic impurities. Thus two phases are separated by a very clearly
defined boundary of coagulated proteins.
The aqueous phase is precipitated and then the DNA could be pelleted after
rounds of purifications.

What is the role of Isopropanol in DNA isolation?


Its main role is to pricipitate the DNA, by engaging with the water molecule
as not giving chance for the DNA to get dissolve in the water.

• What is the role of ethanole in dna isolation?


Ethanol is a dehydrating agent. In DNA isolation it is used for the precipitation
of DNA molecule. When a molecule is to be precipitated it should be
dehydrated as the water molecules forming a film around it prevent their
interaction. In DNA isolation the DNA molecules are dehydrated by ethanol.

• Why Discard the supernatant in early stage of


dna isolation?
Pieces of cell wall are released from the bacteria and are floating around in the
supernatant. These cell wall pieces can inhibit enzyme action on your final DNA, so it
is important to get rid of all of the supernatant and to even invert the tube.

• What is the role of sodium acetate in DNA


extraction?
The function of adding the acetate solution (either sodium acetate or
ammonium acetate) is to raise the salt concentration, and therefore cause
the nucleic acid (here DNA) to precipitate out of solution.
The role of the salt in the protocol is to neutralize the charges on the sugar
phosphate backbone. A commonly used salt is sodium acetate. In solution,
sodium acetate breaks up into Na+ and [CH3COO]-. The positively charged
sodium ions neutralize the negative charge on the PO3- groups on the nucleic
acids, making the molecule far less hydrophilic, and therefore much less
soluble in water.

DNA extraction - why 100% and 70% ethanol is


used?

I added 100% ethanol I have to "incubate" the samples in the freezer (-20) for
about a half an hour, but then I have to to the 70% step, and here I only have
to centrifuge the samples, not "incubate".
Is the 75% step simple an extra purification step to get rid of some 100%
ethanol and other stuff.

Or says to,

70% ethanol to remove salts and small organic molecules, and


resuspended in buffer at a concentration suitable for further
experimentation.

• Why so many centrifuge is used?


Cause to get purify DNA.
 BY PIJUSH SUTRADHAR

Genetic engineering &


Biotechnology Dept.

3rd year 1st semester

• REG.2007432026

 Email: pijushgen@yahoo.com

Pijush_geb07@yahoo.com

 Contact no: 01719232566

SHAHJALAL UNIVERSITY OF SCIENCE AND TECHNOLOGY