Lab 1

Rima Mohamed Akram

Gene elution PCR assay

Insertion Transformation into cloning vector

DNA isolation

1. 2. 3.

Cell lysis DNA separation DNA purification

This process is essential for the destruction of protein structures and allows for release of nucleic acids from the nucleus.

Lysis solution usually contains detergents to solubilize the cell membrane and proteinase K to denature the proteins.

 In

plant or hard tissues like bones or wood, DNA isolation is hard to achieve by lysis solution only; suggest a solution?

freezing in liquid nitrogen and subsequently pulverizing the tissues to a fine powder, followed by homogenization in lysis buffer using a homogenizer.

Lysed samples are mixed with phenol, chloroform, and isoamylalcohol for separation of DNA and protein.

Phenol and chloroform cause proteins to become denatured and become soluble in the organic phase or interphase, while nucleic acids remain in the aqueous phase. Phenol: chloroform mixture is more effective to precipitate proteins than using each solution separately.

Chloroform: isoamyl alcohol is a type of detergent. It binds to protein and lipids of cell membrane and dissolve them. By this it will disrupt the bonds that hold the cell membrane together and cause it to breakdown. It then form complexes with these lipids and proteins, causing them to precipitate out of solution. N.B.: lipids and proteins are non-aqueous compound and DNA/RNA are aqueous compound. The detergent binds to non-aqueous compound. Isoamyl alcohol is added to phenol:chloroform to reduce foaming.

What is the difference between using ethanol or isopropanol for DNA percipitation? Isopropanol percipitates DNA faster, but will also give a high amount of salt percipitate. Ethanol takes longer but provides a higher yield.

ethanol: to precipitate the DNA 30% water: to dissolve residual proteins thus provide more purification since DNA will be found in pellet.
70%

isolation using Fermentas kit 0.8% agarose gel electrophoresis DNA visualization using UV transilluminator
DNA