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How to clone?
Hi there,

I am Mohan studying MSc Drug Design and Biomedical Sciences. In one of our modules we have a course work on designing an experiment, as per the course work details provided we are given a gene of interest which is one of the penta functional gene of an organism and we are asked to work on our method of approach for the course work. I have a clear idea about the PCR technique but before that the procedure is very confusing, about how to isolate a gene of the desired protein from the protein cluster then how to design the primers (I have used some online tools), how to identify the restriction enzymes. we have been given a specific vector which IS pGEX-4T-1, but what is the other alternative if the restriction sites are not available on the gene? A lot of doubts, but when I approach professors each one is giving there own reviews and approaches which are confused among themselfs. Please help me in this.. all the above approaches should be made with the online bioinformatics tools.. thank you.
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A gene can be cloned by following the different steps of the cloning. Inevitably, the process of gene cloning is not a new one. Most of the people and novice biotechnologists know its basic things but to help you in studying, i would suggest to carefully read the following methods:
To start with the cloning process of a gene , DNA has to be separated from the organism which comprises the needed gene.

The separated DNA is purified and then scattered with certain restriction enzyme. Exactly, restriction enzymes are used with regard to cloning. And, they have the capability to produce staggered cuts in certain squences of the Dna, generating scatters with cohesive ends.

Each scattered Dna has a single and stranded sequence of nucleotides to its ends which has the capability to hybridize with Dna which has been scattered with the same restriction enzyme.

Apart from this the fragments of the Dna are incorporated over Plasmids . Exactly, the kind of plasmid proposed to be used for cloning purpose has one restriction site and when it cleaves the said form of enzyme, it generates the similar cohesive ends which exists in the Dna what is about to clone.

The whole process discussed above ultimately line up the Dna fragments and plasmids and in the end form phosphodiesterbonds.

After going through the above cited processes and methods, you need to go through the second step of cloning which begins with incorporating plasmids in the host cells through transformation. Exactly, each of them contains a vaious segment of the Dna from the genuine organism. And, when they are formed together,the cells represent the entire library of the Dna. After that cells could be plated over argar medium and the colony of cells comprising of the needed gene which is to be clone, could be parted away and separated.
Sasa Milosevic
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