Question

Describe in detail how to clone your “gene of interest” into a bacterial plasmid using EcoRI restriction enzyme and how to generate a large quantity of the DNA using competent E. coli. Why do you use competent bacteria for this?

Answer 1

ANS- GENE CLONNING is the process through which the gene of interest is cloned (replicate the copies of gene)by using plasmid which is a circular DNA strand other than chromosome that can replicate independently present in  E.coli bacteria.

• Competent E.coli bacteria is used for the gene clonning because a competent bacteria is having the capability to readily uptake the extra the plasmid from environment and hence there will be large quantity of DNA will be produced.

STEPS FOR GENE CLONNING-

STEP 1-first take a plasmid out of the E.coli cell.and the gene of intreast from the human DNA using E.coli restriction enzyme.

STEP 2-Then restriction enzyme E.coli will act on a specific site on a plasmid caled restriction site and remove a gene from the plasmid DNA leaving a stickey ends behind so that the gene of interest can stick or attach there.

• In both cases it is necessary that E.coli restriction enzyme will be used to cut out the gene from the DNA.

STEP 3-Gene of interest will be added on plasmid DNA and attach with using ligase enzyme.

STEP 4-Now plasmid will be put into the E.coli bacterium through transformation process.here the competent bacteria will need so that it can readily uptake the plasmid DNA carrying gene of interest.

STEP 5-E.coli bacteria plated into agar or any other medium for the clonning, where the E.coli will replicate and produces multiple copies of itself, as the E.coli bacteria multiplies the plasmid DNA carring gene of interest will also be multilied and as a result of that our gene of interest will be increse in large quantity.

STEP 6- Take out the gene of interest from E.coli for further purpose/ experiment.