A single band form is given to intact high quality DNA (small amount of degradation is tolerable).Gels are stained with ethidium bromide to permit photography under UV light.The large molecules are held up while smaller ones move faster causing separation by size. Nucleic acids have negative charge and move from left to right.Generally, 0.7 – 2% gel is considered to be adequate for most of the applications. Gels consist of microscopic pores and are solid (usually agarose or polyacrimide).The percentage and size of the gel to be used must be determined.Sorts the complex mixture of DNA fragments according to size.PCR amplifies the number of fragments of DNA obtained from the restriction digest which are easily separated using gel electrophoresis.The components are then added into a PCR tube and mixedby absorbing the contents with the help of pipette slowly avoiding formation of any bubbles.Before appropriate DNA concentration and establishing a restriction digestion with preferable enzymes, we keep reagent necessary for digestion process on ice.Each restriction enzymes are validated with universal buffers (L, M, H, K, or T (+BSA)) and supplied with recommended buffer.We use restriction endonuclease enzyme to break long nucleotide sequence into smaller fragments for purification or identification process.Visible DNA fibers are removed and suspended in buffer.We then use alcohol precipitation to purify the DNA from the solution.Proteins are removed through organic and non-organic extraction.We then incubate the specimen with detergent to promote cell lysis (frees cellular proteins and DNA).We separate the DNA to be tested from the rest of the cellular material in the nucleus.The probes are labeled with a marker and complementary to the target DNA as a result we can detect one molecule of target in a mixture of millions after hybridization as the reactions are specific. Hybridization is a technique in which a double stranded DNA molecule is formed in between a single stranded DNA probe and a target single stranded DNA. It is based on the principle of transfer of separated DNA fragments to a carrier membrane (usually nitrocellulose) using gel electrophoresis and subsequent identification of specific DNA fragmentsby labelled probe hybridization. The most common and popular membranes are made of nitrocellulose, uncharged nylon positively charged nylon but they are interchangeable depending on the applications. There are different types of membrane, transfer buffer and transfer methods to set up a southern blot. The DNA are then exposed to hybridization analysis allowing bands with sequence resemblance to a labeled probe to be identified. During southern blotting, the DNA fragments are immobilized as a result, the membrane carries a semi-permanent reproduction of the banding pattern of the gel. Southern blotting is a molecular biology technique used for DNA detection, characterization, and quantification.Īn example of RFLP(restriction fragment length polymorphism), southern blotting can be defined as an analytical technique for identifying specific sequences of DNA by separating fragments on a gel and transferring them to a second medium (carrier membrane) on which hybridization testing may be carried out. Southern integrated three innovations to create the Southern blot – restriction endonucleases, gel electrophoresis and blotting through methods.DNA fragments were differentiated using electrophoresis based on size, then transferred to a membrane and hybridized with a radio labeled DNA probe. It was introduced as a technique to detect particular sequence of DNA in DNA samples. Edwin Southern, the inventor of Southern blotting started a trend to his invention after him.
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