RecombinantDNADaredevils

Yen Phan, Jen Masciovecchio, Cristina Johnson, Praj Archaya, 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?