Exam 190826 Flashcards

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1
Q

What’s PEI used for?

A

PEI is a trasnfection reagent. It’s cationic, which lets it bind to anionic cell surface receptors.

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2
Q

Does trypan blue mark viable- or non-viable cells? Explain your reasoning.

A

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.

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3
Q

What can a plaque assay be used for?

A

Plaque assays are used to find the amount of infections / volume added to a cell culture. This is known as the virustiter.

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4
Q

True or false: Insect cell lines Sf9 and sf21 have the same growth conditions as most human cell lines.

A

False. The insect cell lines sf9 and sf21 grow optimally at 26-28 degrees celsius. There’s no need for extra CO2.

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5
Q

Where does HEK and CHO cell lines derive from?

A

HEK = Human embryonic kidney

CHO = Chinese hamster ovaries

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6
Q

What’s a chaotropic agent?

A

Chaotropic agents disrupts the hydrogen bonding ability between water molecules. The disruption of water molecules often affect dissolved molecules’ conformation negatively.

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7
Q

What’s salt fractionation? What salt is commonly used for the fractionation?

A

It’s when you force precipitation of certain molecules by increasing the ionic strength of the solution. The most common salt is (NH4)2SO4

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8
Q

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?

A
  1. Affinity tag.
  2. 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.
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9
Q

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

A

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.

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10
Q

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

A

b. Southern blotting.

Southern blot probes DNA. Northern blot probes RNA.

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11
Q

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. A mechanism by which green fluorescence protein provides visual clues to a protein’s location in the cell.

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12
Q

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

a) plasmids that contain fragments of λ DNA including the cos site

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13
Q

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

A

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

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14
Q

What’s a touchdown PCR?

A

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.

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15
Q

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

A

TEMED + API weren’t added.

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16
Q

These questions concerns troubleshooting of SDS-PAGE. Suggest what could be wrong and how to solve the following problems.

b) The samples do not sink to the bottom of the well of the gel

A

The sample buffer doesn’t contain enough glycerol.

17
Q

These questions concerns troubleshooting of SDS-PAGE. Suggest what could be wrong and how to solve the following problems.

c) The run takes an unusual long time

A

The buffers are too concentrated.

18
Q

Tabell om SDS-PAGE.

A
19
Q

What’s the millimolar extinction coefficient?

A

The millimolar extinction coefficient relates the absorption of UV-light to molecular concentration.

Vice versa, you can figure out the absorption of a molecule by knowing its molar extinction coefficient to its concentration and the distance from the light source.

A = (epsilon) * concentration * distance from light source.

unit of epsilon (molar extinction coefficient): (M^-1/cm)

“Using the Beer–Lambert law it is possible to relate the amount of light absorbed to the concentration of the absorbing molecule.”
- Wiki

20
Q

(4p, CvW) The enzyme-linked immunosorbent assay (ELISA) is commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. There are different variants of the method. Describe the general principles of ELISA and the particular variants DAS-ELISA, TAS-ELISA and competitive ELISA.

A

The general principle of ELISA is similar of the microarray. Instead of measuring RNA abundance, you measure antigen abundance. The abundance is assayed by antibodies binding to it in four different fashions: Directly / Indirectly / Double sandwich / Triple sandwich.

Direct = Antigen coats the plate, antibody is added (conjugated to fluorophore).

Indirect = Antigen coats the plate, primary antibody is used for signal amplification. Secondary antibody carries the fluorophore.

DAS = Double antibody sandwich. You coat your wells with antibodies, add the antigen, add another antibody which fluoresces. There are washes in between the steps.

TAS = Triple antibody sandwich. It’s DAS, but the fluorescence is amplified by a third antibody which carries the fluorophore.

21
Q

a) Roughly, what is the limit of resolution in light microscopy? (1p)

A

0.2 um.

Reason: Airy disc overlap.

22
Q

B) A molecular beacon…
(1) has a stalk and a gene-specific part
(2) has two fluorescent groups
(3) has a fluorescent group and a quencher

A

(3) has a fluorescent group and a quencher

23
Q

A) siRNAs can be used in…
(1) Northern blotting
(2) qPCR
(3) RNA interference

B) A molecular beacon…
(1) has a stalk and a gene-specific part
(2) has two fluorescent groups
(3) has a fluorescent group and a quencher

C) For gene expression quantification, RNAseq…
(1) can only be used for species with sequenced genomes
(2) may be used for species without sequenced genomes, but it is more difficult and results are less reliable
(3) raw data can be analysed on a standard laptop

D) The Tm of a primer…
(1) depends only on the % GC
(2) is the temperature when 90% of the primer molecules are bound to the complementary DNA
(3) is the time it takes for the primer to efficiently bind to the complementary DNA

E) Benjamini-Hochberg…
(1) is a statistical method to certify that you have no false discoveries
(2) is a statistical method of regression, for ranking genes responding to a treatment
(3) is a statistical method to correct for parallel testing by using false discovery rate

F) A RISC complex…
(1) can bind a single stranded short RNA and then induce a translational repression for an mRNA
(2) can bind a single stranded short RNA and then induce the degradation of an mRNA
(3) can bind a single stranded short RNA and an mRNA and then synthesise a new strand of cRNA on the mRNA

A

A) siRNAs can be used in…
(3) RNA interference

B) A molecular beacon…
(3) has a fluorescent group and a quencher

C) For gene expression quantification, RNAseq…
(2) may be used for species without sequenced genomes, but it is more difficult and results are less reliable
(3) raw data can be analysed on a standard laptop

D) The Tm of a primer…
None

E) Benjamini-Hochberg…
(3) is a statistical method to correct for parallel testing by using false discovery rate

F) A RISC complex…
1) can bind a single stranded short RNA and then induce a translational repression for an mRNA
(2) can bind a single stranded short RNA and then induce the degradation of an mRNA

24
Q

Describe how you can make single stranded cDNA from isolated total RNA?, i.e. all the components needed and steps involved all the way up to having the cDNA free in solution. (3p)

A
  1. (Optional) Enrich the RNA which you want to make into cDNA. You could for example enrich mRNA.
  2. Treat RNA with DNAse, incubate, then add EDTA to inactivate the DNAse.
  3. Prepare RNA for cDNA synthesis with the following reagents.
    - DNA polymerase
    - RNAse inhibitor (for stability)
    - DTT (for optimal polymerase activity)
    - Buffer (for optimal ionic strength and pH)
    - dNTPs
    - Primers (OligoDTs / Random / gene-specific for mRNA / all RNA / specific genes)
25
Q

D) How will your choice in (C) be effected by the quality of the isolated RNA, the properties of your target mRNA, which part of the target cDNA you should design your amplicon for, and if you want to use the same cDNA for quantifying mRNA from multiple genes by qRT-PCR. (3p)

A

OligoDTs: Full mRNAs. long mRNAs will not be fully amplified.

Random: Very fragmented cDNA, high yield.

Specific: Very diluted cDNA. ‘

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