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Recombinant
DNA
Daredevils
Yen Phan, Jen
Masciovecchio, Cristina
Johnson, Praj Acharya, Julie
David
Cloning

Cloning is used by scientists to study a particular gene that they
are interested in

Genetic code is universal for all species.
Cut &Paste

First they have to purify the gene. Bacteria and Virus all have
vectors which are used to store DNA information such as
antibiotic resistance gene. The plasmid/vectors are called the
origin of replication. We just use it to clone genes we want.
Through evolution bacteria have created many restriction enzyme
which is protein that catalyze a reaction.

It catalyzes the breaking of phosphodiester bond in a specific
sequence. So basically, it is used to identify the nucleotides of
interest and cut it. Each restriction enzyme cut at specific sites.
The DNA sequence of interest is cut at the same sites.
Vector &Transformation

When mixed together both the vector and DNA joined together
through a hydrogen bond. Ligase, used to bond the okazaki
fragment in lagging strands, is then used to covalently tape it
together.

To defend itself from cutting it's own DNA, the bacteria has a
methylation group in the site where the restriction enzyme can't
cut it. The vector mixed with bacteria and they would take the
vector in normally.

This is called horizontal transfer when the bacteria shared DNA
to make sure that the genes best fit for the environment get
passed to other bacteria. If we stock them with heat and cold. It
will take the vector in at a faster rate.
Selection

Then, the bacteria is placed in a petri dish with food and stuff for
bacteria to reproduce. All the bacteria with or without the vector
will clone and create a colony around it.

This is known as the library where it has millions of different
bacteria with different vectors and different genes stored in it.
Since the bacteria with the vector already have
the antibiotic resistance gene, we want to just have to put
ampicillin, penicillin, or whatever antibacterial drug used to kill
the ones without the vectors. The ones that take in the vector
will live.

Next, the bacteria is placed in a plasmid prep to break up the
vector from the bacteria.
Gel Electrophoresis

Next, the vector is placed in an agarose or a jello bar. It has a
well to put the vectors in and a negative charge on one end
and a positive charge on the other end. DNA is negatively
charged so it will go to the positive charge.

Also, there will be a molecular weight ladder used as a ruler
to mark where the base cut would be in the vector. The band
is where you see the line or where the cut would be. From a
vector with usually about 4,000 bases pairs, you can use the
band and ruler to see how many cuts take place and where
each cut is or how close they are.
Questions



How do we know what parts of
the DNA are reconnected after
the restriction enzymes have
made their cuts?
How do restriction enzymes
know exactly where to cut?
How does some viruses obtain
their DNA methylation group in
the first place? It was not very
clear on that in the video.

How does virus's genes go into
the vector? Through the same
restriction enzyme we used?

How do they know which
antibiotic restriction gene to put
into the vector?

Beside antibiotic restriction
genes, do other genes go into the
vector too?

Is the shorting of the gel
electrophoresis based on the
charges or on the weight?