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Lab 3 DNA Extraction

This document describes the process of extracting genomic DNA from plant cells using the cetyltrimethylammonium bromide (CTAB) protocol. The basic steps involve lysing the cell membrane using a CTAB extraction buffer to release the genomic DNA. Impurities are then separated from the CTAB-DNA complex through chloroform extraction and purification. Finally, the DNA is precipitated from the complex using a precipitation solution containing sodium acetate and ethanol washes, liberating pure genomic DNA.

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Serdar Abdulkerim Gulli
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103 views

Lab 3 DNA Extraction

This document describes the process of extracting genomic DNA from plant cells using the cetyltrimethylammonium bromide (CTAB) protocol. The basic steps involve lysing the cell membrane using a CTAB extraction buffer to release the genomic DNA. Impurities are then separated from the CTAB-DNA complex through chloroform extraction and purification. Finally, the DNA is precipitated from the complex using a precipitation solution containing sodium acetate and ethanol washes, liberating pure genomic DNA.

Uploaded by

Serdar Abdulkerim Gulli
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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DNA extraction Lab 3

The genomic DNA consists of the entire DNA present in a single cell or group of cells.

DNA isolation is a routine procedure to collect DNA for sub-sequent molecular analysis such
as PCR, Sequencing, and gene cloning etc…..

The principle of extraction of genomic DNA that;

1. Organic solvents can degrade and precipitate all components of the cell except nucleic
acids.
1. Only nucleic acids can precipitate by alcohols.

The basic criteria a method of DNA isolation from any sample type should meet include:

1-Efficient extraction.
2-Sufficient amount of DNA extracted for downstream processes.
3-Removal of contaminants.
4-Quality and purity of DNA.

The basic steps involved in all DNA extraction methods consist of the following:

1-Cell lysis. The first step involves disrupting the cell to access the DNA. This can be done
using chemical or physical methods.

2-Lipid removal. Removal or separation of membrane lipids and cell debris. This is usually
done by using detergents such as sodium dodecyl sulfate, and by centrifugation.

3-Deproteinize cell extract. Protein denaturation is done using a protease such as pronase and
proteinase K. Subsequently denatured protein is separated from the cell extract.

4-RNA removal. Removal of RNA is done by adding an RNase, which rapidly degrades
RNA into ribonucleotide subunits. This step is usually optional within most kits.

5-DNA precipitation/aggregation/elution.

Isolation of plant genomic DNA by cetyltrimethylammonium bromide (CTAB) protocol

Plant cells can be lysed with the ionic detergent CTAB, which forms an insoluble complex
with nucleic acids in a low-salt environment. Under these conditions, polysaccharides,
phenolic compounds and other contaminants remain in the supernatant and can be washed
away.
1. Lyses of cell membrane: extraction buffer containing NaCl, EDTA, Tris/HCl and CTAB.
When the cell membrane is exposed to the CTAB extraction buffer, the detergent captures the
lipids and the proteins allowing the release of the genomic DNA. In a specific salt (NaCl)
concentration, the detergent forms an insoluble complex with the nucleic acids. EDTA is
chelating components that among other metals bind magnesium. Magnesium is a cofactor for
DNAase. By binding Mg with EDTA, the activity of present DNAase is decreased. Tris/HCl
gives the solution a pH buffering capacity (a low or high pH damages DNA).

2-Purification: In this step, polysaccharides, phenolic compounds, proteins and other cell
lysates dissolved in the aqueous solution are separated from the CTAB nucleic acid complex.
Under low salt concentration, the contaminants of the nucleic acid complex do not precipitate
and can be removed by extraction of the aqueous solution with chloroform. The chloroform
denatures the proteins and facilitates the separation of the aqueous and organic phases. Once
the nucleic acid complex has been purified, precipitation can be accomplished.

3. Precipitation: In this final stage, the nucleic acid is liberated from the detergent. For this
purpose, the aqueous solution is first treated with a precipitation solution(ethanole)
comprising of Sodium acetate, which precipitates the nucleic acid. Under these conditions, the
detergent, which is more stable in alcohol than in water, can be washed out, while the nucleic
acid precipitates. The successive treatment with 70% ethanol allows an additional purification,
or wash, of the nucleic acid from the remaining salt

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