Volledige tekst van de MBE presentatie van de opleiding biologie en medisch laboratoriumonderzoek. In de presentatie worden de volgende technieken beschreven: Electroporatie / transformatie, qPCR (vs PCR), IC 50 determinatie, Äkta chromatografie. Met deze presentatie is een 10 gehaald
The purpose of electroporation and transformation
During MBE, transformation is used to post a plasmid with the gene of interest in a bacterial cell. This
is done in order to be able to do further research with the plasmid, where the gen of interest is
inserted. We can also make more of the plasmid with this method. The electroporation in a
eukaryotic cell is carried out by placing the gene of interest in a Dicty cell, by means of a plasmid. This
is done to let the gene be expressed, this is used for example to localize the gene in a cell.
Method and technic
Transformation is a genetical alteration of a cell, which is the result of the uptake of exogenous
genetic material from its environment through the cell membrane. Bacterial cells and eukaryotic cells
do not just take up foreign DNA, you need to do chemical transformation or electroporation to make
competent cells. Competent cells are cells that can take up foreign DNA. The chemical
transformation is used for E.coli cells, this is also called heat shock or the calcium chloride method. In
chemical transformation the cell wall of bacteria can be made weak by incubating them at a low
temperature in a buffer with certain salts. When you add the plasmid to the cells, and incubate them
short on 42 degrees celcius, a portion of the DNA will be added in bacteria. Electroporation can be
used for bacterial cells and eukaryotic cells, when they are exposed to an electrical field the
permeability from the cell membrane will be higher. This is the reason why foreign DNA can be
introduced in the cells.
Results and conclusion
There is a change that treated bacteria have died during the transformation. And no matter what
method you use, there will always be many cells who didn’t take the added plasmid. To only keep the
bacteria with the plasmids, we use a selective culture medium. In this case, we added Ampicillin,
when the bacteria don’t make a protein which is resistant to ampicillin, they will die and there won’t
be growth. We only had growth on the plate without ampicillin and a normal E.coli kollonie, this was
the positive control and you expect a lot of growth. On all the other plates we didn’t have any
growth. This could mean that the concentration ampicillin on the plate was to high or the E.coli cells
had died elsewhere during the process. This results means that the transformation in E.coli had
failed. Luckily there where spare kolonies with the plasmid to continue the other experiments. These
are the results from the electroporation, on this pictures you see the dicty cells under a microscope
with a fluorescense filter. You can see the red fluorescence in the cells. This is the expression of the
gene of interrest.
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