Lab 4 Flashcards
Agarose gel components
- Electrophoresis buffer
- Agarose powder
- Intercalating dye
Electrophoresis buffer material
- Tris-borate-EDTA
- Tris-acetate-EDTA
What is needed for one gel?
About 30ml buffer where 0,3 g of agarose is mixed
Why is TAE or TBE buffer used during agarose gel electophoresis?
Provide ions to carry the current and to maintain the pH at a relative constant value
What does the loading buffer contain?
A dense compound that increases the density of the sample and coloured dyes (xylene cyanol and bromophenol blue)
How does the colouring dye xylene cyanol work?
Light blue colour - migrated together with the large fragments
How does the colouring dye bromophenol blue work?
Dark blue - migrates together with the smaller fragments
What is used for optimal resolution during electrophoresis?
10 V/cm electric current
How does the DNA migrate in the gel during electrophoresis?
From neg. to pos. pole with a speed determined on the size: smaller fragments migrate faster
Advantage of indirect investigation - virus neutralisation test?
That AB are longer present in the blood, higher chance for diagnosis of a former virus infection
Disadvantage of virus neutralisation test?
Usually can’t differentiate the maternally or vaccine-derived AB, or seroconversion
When is the sample taken during paired sera investigation of virus neutralisation test?
1st: at the onset of clinical signs
2nd: 10-14 days later
How can the neutralisation be detected?
By the lack of reactions such as CPE, plaque formation or disease in animals
What type of result does the determination of the neutralisation antibodies give?
A serotype-specific result
What is constant virus varying serum dilution method used for?
For AB detection and the determination of the AB-level of the blood sample
How is the virus neutralisation test evaluated?
Virus neutralisation titre is the greatest dilution of the serum where there is 50% CPE
How can the neutralisation titre be determined in case of viruses multiplying in embryonated eggs?
By serial serous dilution mixed with determined quantity of the virus, after inoculating them into embryonated eggs
Where is virus neutralisation test usually preformed?
In a 96-we’ll plastic plate
What is the plastic plate of the virus neutralisation test divided into?
- Serum (assay) part
- Virus control part
- Cell control part
What is serum part used for?
Investigation of the samples
What is done in the cell control part?
Negative control
What is done to titrate?
A serial of tenfold dilution of the virus suspension is prepared and inoculated into several wells of the plate
How to evaluate the titration?
By the nr of the inoculated cell cultures where CPE can be observed
What is prepared during the serum wells?
Serial twofold dilutions of serum samples
How is the virus neutralisation titer calculated?
By finding the highest dilution of the serum where CPE occurrence in at least two of the inoculated cell cultures
What does each of the serum part of the plate contain?
- Cell culture
- Virus (constant)
- Seta (diluted)
- Cell culturing media
What does each well of the virus control part contain?
- Cell culture
- Virus (10 fold dilution)
- Cell culturing media
What does each well of the cell control part contain?
- Cell culture
- Cell culturing media
