Exam 190826 Flashcards
What’s PEI used for?
PEI is a trasnfection reagent. It’s cationic, which lets it bind to anionic cell surface receptors.
Does trypan blue mark viable- or non-viable cells? Explain your reasoning.
Trypan blue colors non-viable cells due to their cell walls getting permeabilized. This is better than staining live clls, since the dye may interfere with some molecular machinery.
What can a plaque assay be used for?
Plaque assays are used to find the amount of infections / volume added to a cell culture. This is known as the virustiter.
True or false: Insect cell lines Sf9 and sf21 have the same growth conditions as most human cell lines.
False. The insect cell lines sf9 and sf21 grow optimally at 26-28 degrees celsius. There’s no need for extra CO2.
Where does HEK and CHO cell lines derive from?
HEK = Human embryonic kidney
CHO = Chinese hamster ovaries
What’s a chaotropic agent?
Chaotropic agents disrupts the hydrogen bonding ability between water molecules. The disruption of water molecules often affect dissolved molecules’ conformation negatively.
What’s salt fractionation? What salt is commonly used for the fractionation?
It’s when you force precipitation of certain molecules by increasing the ionic strength of the solution. The most common salt is (NH4)2SO4
Describe two modifications of the cDNA (coding for your protein) in order to facilitate protein production. In which of the stages that you describe under a. do you have to do these modifications?
- Affinity tag.
- Solubility factors. You may want to change an amino acid residue which in turn affects the protein conformity, solubility, incliniation to form inclusion bodies, where it will be compartmentalised.
Gibson Assembly efficiently joins multiple overlapping DNA fragments in a single-tube isothermal reaction. The Gibson Assembly method requires different enzymatic activities.
These are:
a) Protease, reverse transcriptase and RNAse H
b) Exonuclease, phosphatase and DNAse I
c) Exonuclease, DNA polymerase and DNA ligase
d) Exonuclease, RNA polymerase and DNA ligase
e) Endonuclease, DNA polymerase and DNA ligase
c) Exonuclease, DNA polymerase and DNA ligase
Exonuclease is required for 5’ digestion.
From the remaining options, you wouldn’t need DNAse or RNApol, thus, c) must be correct.
The technique of _______________________ is used to transfer DNA from an agarose gel to a membrane.
a. RT-PCR
b. Southern blotting
c. restriction mapping
d. Western blotting
e. Northern blotting
b. Southern blotting.
Southern blot probes DNA. Northern blot probes RNA.
Fluorescence resonance energy transfer (FRET) refers to
a. a process by which radiationless transfer of energy occurs from an excited state fluorophore to a second chromophore in close proximity
b. a mechanism by which green fluorescence protein provides visual clues to a protein’s location in the cell
c. interaction between components in the yeast two hybrid system that results in gene expression
d. different visual responses from different primers in multiplex PCR
e. a change in visual appearance of DNA when it interacts with a protein
a. A mechanism by which green fluorescence protein provides visual clues to a protein’s location in the cell.
Cosmid vectors are
a) plasmids that contain fragment of λ DNA including the cos site
b) phages that lack cos site
c) plasmids that have no selection marker
d) cryptic plasmids
e) mini chromosomes
a) plasmids that contain fragments of λ DNA including the cos site
There are two common methods of end-labeling of a DNA fragment: the “fill-in” reaction and the “kinase” reaction. The fill-in reaction uses the __________domain of Escherichia coli DNA polymerase I.
a. Polynucleotide kinase
b. Terminal deoxynucleotidyl transferase
c. 3´–> 5´ Exonuclease
d. 5´–> 3´ Exonuclease
e. 5’ –> 3’ DNA polymerase
e. 5’ –> 3’ DNA polymerase
Dubbelkolla om tid finnes
https://www.thermofisher.com/se/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/methods-labeling-nucleic-acids.html
What’s a touchdown PCR?
It’s when you run your intitial PCR cycles at a T>Tm primers. The reasoning is that the primers can only bind to their most complementary domains. Unspecific bands are removed.
These questions concerns troubleshooting of SDS-PAGE. Suggest what could be wrong and how to solve the following problems.
a) The gel does not polymerize
TEMED + API weren’t added.
