Hi,
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!
Happy cloning!!!
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