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What Does DNA Extraction Involve

DNA extraction involves breaking open cells to release DNA, separating DNA from other cellular components, precipitating DNA with alcohol, and cleaning and analyzing the extracted DNA. Specifically, cells are lysed using detergents and salts to disrupt membranes and release DNA. Proteases and filtering remove proteins and debris. DNA precipitates out of solution when alcohol is added due to its insolubility in alcohol. The extracted DNA can then be purified, quantified, and evaluated for quality before being used in downstream applications like PCR, sequencing, and cloning.

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Suneel Kumar Jaipal SK
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59 views2 pages

What Does DNA Extraction Involve

DNA extraction involves breaking open cells to release DNA, separating DNA from other cellular components, precipitating DNA with alcohol, and cleaning and analyzing the extracted DNA. Specifically, cells are lysed using detergents and salts to disrupt membranes and release DNA. Proteases and filtering remove proteins and debris. DNA precipitates out of solution when alcohol is added due to its insolubility in alcohol. The extracted DNA can then be purified, quantified, and evaluated for quality before being used in downstream applications like PCR, sequencing, and cloning.

Uploaded by

Suneel Kumar Jaipal SK
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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What does DNA extraction involve?

Step 1. Breaking cells open to release the DNA


The cells in a sample are separated from each other, often by a physical means such as
grinding or vortexing, and put into a solution containing salt. The positively charged
sodium ions in the salt help protect the negatively charged phosphate groups that run along
the backbone of the DNA.
A detergent is then added. The detergent breaks down the lipids in
the cell membrane and nuclei. DNA is released as these membranes are disrupted.
Step 2. Separating DNA from proteins and other cellular debris
To get a clean sample of DNA, it’s necessary to remove as much of the cellular debris as
possible. This can be done by a variety of methods. Often a protease ( protein enzyme) is
added to degrade DNA-associated proteins and other cellular proteins. Alternatively, some of
the cellular debris can be removed by filtering the sample.
Step 3. Precipitating the DNA with an alcohol
Finally, ice-cold alcohol (either ethanol or isopropanol) is carefully added to the DNA
sample. DNA is soluble in water but insoluble in the presence of salt and alcohol. By gently
stirring the alcohol layer with a sterile pipette, a precipitate becomes visible and can be
spooled out. If there is lots of DNA, you may see a stringy, white precipitate.
Step 4. Cleaning the DNA
The DNA sample can now be further purified (cleaned). It is then resuspended in a
slightly alkaline buffer and ready to use.
Step 5. Confirming the presence and quality of the DNA
For further lab work, it is important to know the concentration and quality of the DNA.
Optical density readings taken by a spectrophotometer can be used to determine the
concentration and purity of DNA in a sample. Alternatively, gel electrophoresis can be used
to show the presence of DNA in your sample and give an indication of its quality.
What can this DNA be used for?
Once extracted, DNA can be used for molecular analyses including PCR, electrophoresis,
sequencing, fingerprinting and cloning.

•Cells 10ul of cell culture 100 °C in water in PCR machine for 5 minutes

•Tissues (100 % formamide, heat 95 and 72°C 30 times prior to PCR. Panaccio et al., 2003

•Plant (seed) (drilling out a sample from the seed, adding NaOH, heating in a microwave oven and
neutralizing with Tris-HCl

PROCEDURE

• Cell Lysis Buffer - lyse cell membrane, nuclei are intact, pellet nuclei.

• Resuspend nuclei, add Sodium Dodecly Sulfate (SDS), Proteinase K. Lyse nuclear membrane and digest
protein.

• DNA released into solution is extracted with phenolchloroform to remove proteinaceous material.
• DNA is precipitated from the aqueous layer by the additional of ice cold 95% ethanol and salt

• Precipitated DNA is washed with 70% ethanol, dried under vacuum and resuspended in TE buffer.

REAGENTS

• Cell Lysis Buffer - Non-ionic detergent, Salt, Buffer, EDTA designed to lyse outer cell membrane of
blood and epithelial cells, but will not break down nuclear membrane.

• EDTA (Ethylenediaminetetraacetic disodium salt) is a chelating agent of divalent cations such as Mg2+.
Mg2+is a cofactor for Dnase nucleases. If the Mg2+is bound up by EDTA, nucleases are inactivated.

• Proteinase K - it is usual to remove most of the protein by digesting with proteolytic enzymes such as
Pronase or proteinase K, which are active against a broad spectrum of native proteins, before extracting
with organic solvents. Protienase K is approximately 10 fold more active on denatured protein. Proteins
can be denatured by SDS or by heat.

• Phenol/Chlorform - The standard way to remove proteins from nucleic acids solutions is to extract
once with phenol, once with a 1:1 mixture of phenol and chloroform, and once with chloroform. This
procedure takes advantage of the fact that deproteinization is more efficient when two different organic
solvents are used instead of one.

• Also, the final extraction with chloroform removes any lingering traces of phenol from the nucleic acid
preparation.

• Phenol is highly corrosive and can cause severe burns.

Concentrating DNA Alcohol Precipitation


• The four critical variables are the purity of the DNA, its molecular weight, its concentration, and the
speed at which it is pelleted.

• DNA a concentrations as low as 20 ng/ml will form a precipitate that can be quantitatively recovered.

• Typically 2 volumes of ice cold ethanol are added to precipitate the DNA.

• Solutes that may be trapped in the precipitate may be removed by washing the DNA pellet with a
solution of 70% ethanol. To make certain that no DNA is lost during washing, add 70% ethanol until the
tube is 2/3 full. Vortex briefly, and recentrifuge. After the 70% ethanol wash, the pellet does not adhere
tightly to the wall of thetube, so great care must be taken when removing the supernatant.

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