Wish you a very happy day! Let all you researches result only in success! J
You know, I’m learning cloning, wow, rhyming it is, learning – cloning!
Like this rhythm, cloning is also fun! Yes, fun, interesting! Let me share with you, what I know about cloning.
The first step of any cloning experiment would be selecting a vector. Now, I’m learning cloning using plasmid as a vector.
So, you have to choose a vector for cloning at first. Vector is nothing; it’s just a plasmid DNA into which we can incorporate our gene of interest and transform the vector into a host which will produce the protein coded by our gene of interest.
Vector need not be only p-DNA, viruses could also be used as vector. But, here, we are going to have an overview of cloning using Plasmid vector.
Okay, we had selected the vector, let us assume. What next? We have to look at our gene of interest now. We have to get the sequence of our gene of interest – it’s available in databases like NCBI.
Then, we have to isolate our gene of interest from the total genome of the organism. If it I prokaryote then you can just easily get the total genomic DNA and do a PCR (polymerase chain reaction) for amplifying your gene of interest.
If it is going to be an eukaryotic gene, then you must go in reverse. You must get the total RNA, then from that you have to isolate the mRNA, then from the mRNA you have to get your amplified gene of interest.
Before doing PCR, you must design Primer. The primer must have the restriction sequences at the end, so that the restriction sequence will also get inserted with your gene of interest at the ends.
Then after PCR, the gene f interest could be inserted into the vector just by restricting both your gene of interest and vector. They both will have complementary cohesive ends, so that they could hybridize with each other.
Of course, here there will be nick, but, we need not worry about the nick, once we transform this vector with gene of interest into our host – say E.Coli DH5 alpha, the host mechanism will take care of the nick.
E.coli DH5 alpha is a special strain of E.coli in which the defense mechanism against the foreign gene is knocked out, so that we could transform our gene into it and ask the E.coli to produce our protein, with out any problem of defense action.
This is just an overview of Cloning. There are several things to explain than this.
I’m happy that I know how to clone now!