DNA extraction Flashcards
The use of DNA for analysis usually requires that it is
isolated and purified to a certain extent.
In all methods for the separation of DNA from cellular components, there are three main stages:
- Cell Disruption or lysis.
- Removal of contaminants (RNA and proteins).
- Recovery of DNA
1. Cell Disruption or lysis
The gentlest possible method of cell rupture
is used to prevent the DNA from fragmenting from mechanical shearing.
1. Cell Disruption or lysis
The cell membrane should be solubilized using
detergent (lysis solution)
Cell Disruption or lysis
Cell walls, if present, should be….
digested enzymatically
(e.g. lysozyme treatment)
To maintain DNA integrity cell disruption (& most following steps)
Be done in the presence of EDTA as
EDTA chelate Mg+2 ions needed for enzymes that degrade DNA termed DNase
To maintain DNA integrity cell disruption (& most following steps)
Performed at the tempreture
4°C
To maintain DNA integrity cell disruption (& most following steps)
Autoclave used glassware
to destroy DNase activity
Removal of contaminants (RNA and proteins)
After release of nucleic acids from the cells, RNA can be removed by treatment with
ribonuclease (RNase)
Removal of contaminants (RNA and proteins)
The other major contaminant, proteins, is removed by shaking the solution gently with a
phenol/chloroform mixture
1 denature proteins
2 centrifugation
Removal of contaminants (RNA and proteins)
The aqueous solution (that contains DNA) is recovered and
deproteinized repeatedly, until no more material is seen at the interface.
Recovery of DNA
Finally, the deproteinized DNA preparation is mixed with two volumes of
absolute ethanol (alcohol), and the DNA allowed to precipitate out of solution (as DNA pellets) in a freezer, then centrifuge
Recovery of DNA
After centrifugation, the DNA pellet is
re-dissolved in a buffer containing EDTA
Most of the current reagents used in molecular biology and the most common techniques can now be found
in kit form or can be automated
