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Molecular Cloning
Nucleic Acid Fractination: DNA and RNA should be purified from tissues or cells in for their identification, characterization or manipulation. Nucleic acids are tightly associated with proteins.
After lysis of the cells the mixture is extracted with phenol and/or iso-amyl alcohal and choloform mixture. The proteins are precipitated and rmain at the interphase, and necleic acids are in aquous phase.
After three to four time extraction as described above, the aquous phase is free of any protein. The DNA from the aquous phase can be precipitated by 70% ethanol in presence of sodium acetate salt at low temperature.
Alternatively, cellular extract can be subjected to proteinase K digestion to get rid of all proteins.
To get DNA only from the nucleic acid mixture, the samples are treated with DNase-free Rnase to get rid of RNA.
To isolate RNA, the mixture can be treated with DNase. In order to keep the nucleic acids intact, nuclease inhibitors like chelating agents and specific inhibitors are used during isolation and extraction proceedures. Chromatography: Hydroxyapetite Chromatography
Sourthern Blotting:
Run the DNA on agarose gel and denature the DNA bands with 0.5M NaOH Transfer the DNA bands to Nitrocellulose membrane by blotting method orelectrotransfer Vaccum dry the nitrocellulose membrane at 80 degree Celsius to permanently fix the DNA onto NC membrane Put the membrane in hybidization buffer containing radioactive probe followed by washing and exposure of NC to X-ray film.
DNA seperation by Ultracentrifugation: Sepearation of single stranded to double stranded DNA (singlr strended is a bit denser), plasmid DNA from genomic DNA, Satellite DNA from Chromosomal DNA.
Supercoiled DNA: Some of the circular DNAs have peculiar Supercoiled or supertwisted form some times referred as tertiary structure of DNA. Linking Number (L): The number of times that one DNA strand winds around the other. Twist: (T): Number of complete revolution that one polynucleotide strand makes about the duplex axis. Writhing Number: Number of turns that a duplex axis makes about the superhelix axis.
Topoisomerases:
These enzymes controle the supercoiling of DNA in living systems to provide the correct topological state of DNA.
Type-I topoisomerasees: Act by creating a transient single strand breaks in DNA and then sealing it. Type II topoisomerases: Also known as DNA gyrases, they act by cutting both strands of a duplex, passing the duplex through the break and resealing. Thus they catalyse the stepwise negative supercoiling of DNA at the expense of ATP hydrolysis.