Make the Sample:
Obtain a biological sample containing the desired DNA (for example, cells or tissue).
Fill a microcentrifuge tube halfway with the sample.
Lysis of Cells:
To the sample, add an adequate volume of cell lysis buffer. Detergents are commonly used in lysis buffer to break open cell membranes.
To digest proteins, use proteinase K, and to digest RNA, use RNase. Incubate the mixture for a specific time (typically 30 minutes to 1 hour) at an acceptable temperature (commonly about 37°C).
Extraction of Phenol from Chloroform:
To the lysate, add an equal volume of phenol:chloroform.
Vortexing completely mixes the solution.The mixture should be centrifuged. This separates the organic (bottom) phase from the aqueous (top) phase, which contains DNA.
Transfer the aqueous phase to a fresh tube with care.
DNA precipitation:
To precipitate the DNA, add cold isopropanol or ethanol to the aqueous phase.
Gently invert the tube to mix, then incubate for at least 30 minutes at -20°C or -80°C.
To pellet the DNA, centrifuge the tube.
Cleaning the DNA Pellet:
Remove the supernatant and thoroughly wash the DNA pellet with 70% ethanol.
Centrifuge once more to eliminate any leftover ethanol.
DNA Reanimation:
Allow the DNA pellet to air-dry for a few minutes.
In TE buffer or distilled water, resuspend the DNA pellet. The TE buffer contributes to the stability of the DNA.
Quantification and archiving:
Using a spectrophotometer or other means, quantify the isolated DNA.
For long-term storage, store the DNA at -20°C or -80°C.
DNA isolation is a laborious and necessary process in molecular biology laboratories, serving as the first step in a variety of downstream applications. The technique begins with the collecting of a biological sample and includes precise procedures such as cell lysis, phenol: the chloroform extraction, and DNA precipitation. These procedures effectively extract DNA from other biological components, assuring the purity of the DNA. The successive washing and resuspension procedures help to remove contaminants while preserving DNA integrity. The quantification of extracted DNA offers important information about its concentration and purity, which guides researchers in following investigations. Storing the DNA at low temperatures ensures its long-term stability, allowing it to be used in a variety of genetic research such as PCR, sequencing, and genetic engineering.
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