Quicktests 2018 Flashcards
What is the advantages and disadvantages of confocal microscopy?
Advantages: - higher resolution - higher contrast - 3D reconstructions - only one level is detected Disadvantage: - low resolution - much energy - dangerous (laser)
What is the principle of 2-photon microscopy and what are advantages / disadvantages compared to confocal microscopy
- 2 long wavelengths
- deeper tissue penetration
- less damaging photobleaching
- flexibility in detection geometry
- it is cheaper than konfokal
- easier to handle
- less health risk
Which variables are there in an experiment? Describe this and refer to an example!
- independent variable: the element of the experiment that is actively affected, e.g. pouring the flower -> genotype -> mutant genes
- dependent variable: variables affected by variation of the independent variable -> are measured, e.g. flower growth, climbing behavior; functionality of the synapse
- disturbance variable: e.g. Temperature, light, color, pheromones, day / season
- positive Control: Wild-type, e.g. should climb -> sets baseline
- negative control: mutant, broke something in the synapse -> difficulty climbing
Name the main criteria of a good experiment, their meaning and how they can be achieved! (= experimental design)
The main criteria Objectivity: - carry out various investigations - blind tests - create clear criteria Reliability: reliability, reproducibility - repetitions (3 biological replicates) - test other equivalent method if the same result Validity: validity, accuracy - good experimental design, exclusion of disturbing variables - good starting hypothesis Minor criteria - Economics - usefulness - Ethics - Comparability and standardization
What is the meaning of resolution and can glutamate receptor fields and SV be resolved at 488 nm with an NA of 1?
Distance that allows 2 points to be considered separated
d= lambda/2xNA = 488 nm/ 2x1 = 244 nm
only glutamate receptor can be resolved, SV not
Which three different mutation types are there? (LOF/GOF)
Zero mutant -> LOF no protein hypomorphic mutant -> LOF little protein hypermorphic mutant -> GOF too much protein
What is a polite experiment?
Preliminary tests with reduced number (s) to determine the best experiment conditions + to determine sources of error
e. g. Climb assay
- period of time
- how high animals climb
- size of the test group
- Variance of the test group
- Sexes difference
- Age of the animals
- time of day
Brain stanings - what is PFA and NGS and what is it used for? What do you use for mounting your samples?
PFA: cross-linking of proteins to improve stability, permeability steps degradation
NGS: blockage of unspecific binding site
Vectashield: inhibits photobleaching, ideal refractive index
What is GcAMP?
Fusion protein from GFP, M13 and calmodulin
- also called GECI, is used in in vivo studies of postsynaptic activity
- Ca2 + flows into the cell, binding it to calodulin, which leads to a conformational change of the calmodulin M13 complex and GFP. This is detected as fluorescence.
How does an immunofluorescence assay function and what is an antibody sandwich? (Explain first and second antibody)
Dissection -> Fixation -> Washing -> Permeabilization and blocking -> Primary Antibody incubation (they bind specific to the antigen of interest) -> washing -> Secondary Antibody incubation (they bind to the first specific antibody and contains fluorophones) -> washing -> mounting -> imaging -> analysis
What is GFP, from which organism is it derived and what is it used for in genetic?
Green fluorescent protein, comes from jelly fish;
to label specific proteins of interest
to recognize the location structure specific proteins of interest and finally to define the function
How does the mechanism of RNAi function?
Rebecca: RNA interference is a mechanism for posttranscriptional gene silencing.
The Dicer cleaves dsRNA resulting in siRNA (short interfering RNA) of 21 to 23 nt. The antisense strand of the siRNA is incorporated into the RISC complex (ribonucleoprotein complex) and guides it to the target mRNA by complementary base pairing. Argonaut proteins cleave the mRNA. This leads to degradation.
In Short-term Memory test, why do we need to train two separate groups of flies reciprocally, that is, one group received OCT (with shock) and MCH (without shock), and the other group received MCH (with shock) and OCT without shock?
- to compensate for preference / repulsion
- Flies are usually distributed equally on OCT / MCH, so only the compensation is checked
