Restriction Cleavage and Gel Electrophoresis, DNA Moelcule, Elctrophoresis, Ethidium Bromide,Hypothetical DNA, Gel Calibrated

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Chimeric DNA Molecular Probes and Gene Libraries Chimeric DNA Molecular Probes and Gene Libraries Introduction Restriction Enzymes for Cloning Technique of Restriction Mapping Restriction Clevage and Gel Electrophoresis Construction of a Restriction Map Use of Partial Digests end Labelling and Hybridization in Restriction Mapping Construction of Chimeric DNA Cloning of Plasmid Vectors Directional Cloning of DNA Fragments with Heterologus Protruding Termini Adding Poly dA at 3' Ends of DNA Clone Blunt End Ligation by T4 DNA Ligase Cloning in Phage λ and Cosmid Vectors Cloning in phage λ Vectors Cloning in Cosmid Vectors Hosts for Cloning Vectors Bacteria as Hosts Eukaryotes as Hosts Molecular Probes Preparation of Probes Genomic DNA Probes

Home >> Biotechnology and Genomics >> Chimeric DNA Molecular Probes and Gene Libraries >> Restriction Cleavage and Gel Electrophoresis

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Restriction cleavage and gel electrophoresis

If we take a particular DNA molecule or DNA sample, digest it with a specific restriction enzyme and then subject the sample to gel electrophoresis (a technique in which DNA digest is loaded on gel slab and the DNA fragments move under the influenoe of an electric current), we will notice a series of bands on the gel slab or cylinder.

Cleavage of DNA by two restriction endonucleases A and B into fragments which can be separated by agarpose gel electrophoresis

Cleavage of DNA by Two Restriction Endonucleases A and B into Fragements Which Can Be separated by Agarose gel Electrophoesis

The bands can be observed under ultraviolet light after staining with ethidium bromide. The position of different bands will depend on DNA fragment size, such that smaller the fragment, more rapidly will it move, and longer the fragment, more slowly will it move.

It will mean that the fragment away form the loading site will be smaller and those close to the loading site will represent longer DNA fragments. The gel can be calibrated by using a mixture of DNA fragments of known lengths, so that the positions of the bands of known lengths on this standard gel can be compared with the positions of bands in the experimental DNA digest.

The fragment length for each band of DNA digest can thus be determined sometimes using a computer device. we have shown the results of digestion of 5000 bp long hypothetical DNA molecule digested separately by two enzymes A and B.

The enzyme A cleaves the DNA into four fragments of lengths 2100, ]400, 1000 and 500bp, while the enzyme B cleaves it into three fragments of lengths. 2500, 1300, 1200bp. These data with some additional experiments, can be used to generate a restriction map as shown in the next section.

Technique of double digests to determine cleavage positions of DNA due to one wnzyme with respect to those due to another enzyme (the two enzymes are A and B);

The site of cleavage by enzymes A and B in of the four fragments

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