Lab4 Flashcards
(41 cards)
When is Agarose gel electrophoresis used?
To check wheter the PCR generated virus-specific amplification product, i.e the sample, is positive for the tested virus.
Why is Agarose gel electrophoresis used?
For size separation and visualization of the PCR products.
How is the amplified DNA molecules separated?
By applying an electric current to move the negatively charged molecules through an agarose mix.
How can the DNA produced in the PCR be identified? (by the agarose G.E)
Comparing the size of the product amplified in the sample to the positive control and the molecule weight marker.
What are the components of the Agarose gel?
- electrophoresis buffer
- agarose powder
- intercalating dye
Electrophoresis buffer contains?
Ions that can carry the electric current through the gel.
Usually Tris-acetate-EDTA (TAE, contains the salts of acetic acid) or tris-borate-EDTA (TBE, contains the salts of boric acid) is used.
What type of molecule is the Agarose?
synthetic polysaccharide
What are the characteristics of the gel made from purified agarose?
relatively large pore size, making them appropriate for separation of DNA fragments larger than 100 base pairs.
Difference between shorter and longer molecules in the agarose gel, at the same voltage:
Shorter molecules move faster and therefore travel further than longer molecules in the same timer interval. this is because shorter molecules migrate more easily trough the pores of the gel.
Gel concentration for short and long DNA products
- For shorter DNA products (100-300 bp) more concetrated gel is needed (1.5-2%).
- for longer DNA products (>800 bp) less concentrated gel is needed (0.5,1%)
How can DNA be visualized in the agarose gel?
by using an intercalating dye (mixed in the gel), which fluoresces under ultraviolet light.
What types of buffers are used in the loading of the PCR products?
TAE and TBE
What is the functions of the TAE and TBE buffers?
provide ions that carry the current, and to maintain the pH at a relatively constant value.
What does the Loading buffer contain?
A dense compound (e.g sucrose), that increases the density of the sample so that the DNA sinks to the bottom of the well.
What are the color dyes used in the loading? and functions
Xylene Cyanol and Bromophenol blue, used to visualise the progress of the electrophoresis, as they migrate together with the PCR products in the gel.
- Xylene cyanol (light blue color) migrates together with the large fragments
- Bromophenol blue (dark blue) migrates together with the smaller fragments.
What current is used for optimal resolution in electrophoresis?
10 V/cm electric current.
- the distance in cm refers to the distance between electrodes.
What happens during electrophoresis?
the negatively charged DNA migrates in the gel from the negative pole to the positive pole
After the end of the electrophoresis, the gel is viewed with=
UV transilluminator.
What is a molecule weight marker?
mixture of different size DNA fragments
What happens during Evaluation?
The size of the amplified DNA products are compared to a molecule weight marker
How can we declare a PCR sample positive?
The size of the DNA product amplified mys be what is expected by the position of the primers and the same as the size of the product in the positive control.
How are the viruses investigated indirectly?
the detection of the antibodies produced by the immune system provides information about the virus.
What is the advantage of the indirect virus investigation?
- is that antibodies are present for a longer period in the blood.
- therefore infections that occurred weeks, months or even years before can be investigated.
- there is a higher chance of diagnosis of a former virus infection
What are the disadvantage of the indirect virus investigation?
- the methods used usually cannot differentiate the maternally or vaccine-derived antibodies, or seroconversion.
- therefore in the interpretation of the result, maternal immunity and immunisation should be taken into consideration.
