Plasmid mapping - Essay Example

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Plasmid mapping Introduction: Plasmids are the extrachromosomal DNA molecules, and are mostly double –stranded, circular and covalently closed molecules, varying in size from 1 kb to 200 kb. (Sambrook and Russell 2001). They are found in many bacterial species…
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Download file to see previous pages This DNA is now called as recombinant DNA. These vectors replicate inside the host cell along with the inserted DNA. These vectors are of two types: expression vectors (expression of the cloned gene to give the desired protein) and cloning vectors (produce millions of copies of cloned DNA). (Sambrook and Russell 2001). Restriction endonucleases are the enzymes that cut the DNA at the specific sequences. There are about 200 different restriction enzymes. (Brown 1995). The most common restriction endonucleases are EcoR1, BamH 1 and Pst1. All these restriction enzymes have sticky ends. The recognition sites for these restriction enzymes are as follows: EcoRI recognition site = G|AATTC Bam H1 recognition site = G|GATCC C TTAA |G C C T A GIG and Pst 1 recognition site = CTGCAIG GIACGTC (Siwach and Singh 2007). The pieces of DNA that remain after the digestion with the restriction enzymes are called as restriction fragments. Each restriction enzyme has a unique code and it cuts the DNA into fragments with either sticky or blunt ends. A restriction map gives us the location where the restriction enzyme cuts the DNA. This restriction digestion is used for two purposes: Restriction mapping and specific DNA cleavage for the production of new constructs. The restriction mapping is used to identify the plasmids. The number of DNA fragments and the size of the DNA fragments depend upon the action of the restriction enzyme. These DNA fragments thus obtained are separated using the Agarose gel electrophoresis. Gel electrophoresis is the most powerful technique for separating the biomolecules. The DNA are negatively charged particles that are attracted towards the opposite charge under the influence of electric field. Here the agarose gel is the solid matrix. The solid matrix controls the rate of migration of the molecules based on the size of the particles and the concentration of the gel. The buffer is a mixture of organic and inorganic salts that helps to conduct the electric current between the positive and negative terminals. To visualize the DNA bands present in the gel, stains such as methylene blue and ethidium bromide are used. In our experiment we use ethidium bromide that fluorescence’s under the UV light. Ethidium bromide intercalates between the base pairs of DNA and fluorescence when exposed to the light of 250 – 300 nm. (Sambrook and Russell 2001). Materials and method: The materials are the same as mentioned in the practical handbook. Restriction mapping consists of three important steps. They are restriction enzyme digestion, agarose gel preparation and sample loading. 1) Restriction Enzyme digestion: The unknown plasmid sample is taken and they are digested using the restriction enzyme. The standard concentration of the plasmid DNA is 1 ?g/ 5 ?l. In order to standardize the plasmid and to enhance the enzyme reaction, 2 ?l of enzyme buffer is added to the digest. 1 ?l of the enzyme is added to the sample. The volume of the digest is made upto 20 ?l using the sterile water. The composition of the digest is as follows: For this Restriction digestion, BameH1,Pst1 and EcoR1 restriction enzymes were used. The final volume of each restriction digest was 20 microlitres. ? Hind3 (Marker)-Distance moved in the gel mm Plasmid DNA Enzyme 10Xenzyme buffer Sterile water Total EcoR1 5µl 1µl 2µl 12µl 20µl Pst1 5µl 1µl 2 ...Download file to see next pagesRead More
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