BTE 204: Fundamentals of Genetic Engineering Sadia Sayed

How to manipulate the gene? Target a gene: Gene of Interest (GOI) Purify DNA from living cell Flow chart of Genetic Engineering Manipulation of purified DNA Introduction of DNA into living cell Harvesting product of interest

phage DNA will be needed if a phage cloning vector is to be used. The genetic engineer will, at different times, need to prepare at least three distinct kindsof DNA. total cell DNA (consists of the genomic DNA of the organism along with any additional DNA molecules,such as plasmids, that are present). plasmid DNA phage DNA will be needed if a phage cloning vector is to be used.

Purification of DNA from Living cells 4

Growing and harvesting a bacterial culture 5

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Growing and harvesting a bacterial culture Lysozyme EDTA: removes magnesium ions that is required for integrity of the cell membrane and also chelates MgCl2 and thereby inhibits Dnase. SDS/CTAB/Lauryl Sarcosinate: remember detergents wash lipid substances! NaOH: causes hydrolysis of the membrane (alkaline lysis) NaCl: causes the cell to rupture 7

Purification of DNA from cell extract (Get rid of protein!) 1:1 Phenol Chloroform mixture: The organic layers captures the protein. The aquous layer has the DNA. DNA is charged negatively, so forms hydrogen bond with water. Also protease can be added to degrade protein contanmination. 8

Precipitate DNA from aqueous layer Alcohol addition: Isopropanol or ethanol addition causes the polarity of the solution to change. So DNA precipitates! 9

CTAB method of DNA isolation

DNA purification by Guanidium thiocyanate method

Measurement of DNA concentration 260/280 nm Absorbance: 1.8 for pure DNA; A260 = 50µg of dsDNA/ml 12

Purification of Plasmid DNA Separation on the basis of Size: Gel electrophoresis; supercoiled will migrate towards negative voltage much faster than the open circular . 13

Purification of Plasmid DNA: Steps Lysis of the cell: Lysis solution 1 14

Purification of Plasmid DNA: Steps Lysis of the genomic DNA: Lysis solution 2 and 3; Alkaline denaturation Apply NaOH and then glacial acetic acid 15

Purification of Plasmid DNA: Steps Density gradient centrifugation: Alternative to Phenol-chloroform wash Buoyant density of DNA is 1.7g/cm3 16

Purification of Plasmid DNA: Steps 18

Plasmid amplification

How do we view DNA? Agents that bind to DNA and also illuminates after UV exposure: EtBr (Intercalating agent) But the DNA after EtBr exposure is not usable without column purification 20

Prepation of bacteriophage DNA

Preparation of λ phage DNA Inducers of Lysogeny and lytic: cI protein= lysogeny Balance between bacteria and phage is important 22

Preparation of Bacteriophage DNA Inducers of Lysogeny and lytic: cI protein= lysogeny Balance between bacteria and phage is important 23

Preparation of Bacteriophage DNA Collection of phage particle in PEG 24

Preparation of M13 DNA 25