question with Cell bio lab (PCR) want to prepare it for midterm. 1. What did you
ID: 201735 • Letter: Q
Question
question with Cell bio lab (PCR)
want to prepare it for midterm.
1. What did you design primers to amplify? Why, what is the point? 2. What is autophagy? What role does autop cells? Audophts self hagy have in the normal function of eukaryotic hat is the purpose of using lor phase els when perfórming an experimenti? at is the purpose of starving cells or treating them with HO proteins are part of the initial complex? Why could it be difficult to express S. What is the first step in th e assembly of the autophagosome? How is TOR in volved? what 6. Tetrahymena genes in yeast? How can the problem be dealt withi? ular micrometer at 10X and find that the distance between divisions s 10 m, what is the size of a cell that measures 5 divisions under the oil immersion lens he 10X What adjustments do you need to make when you are examining a cell usingt objective and then want to examine the same cell under the 40X phase object 8. 9. The Cell Sample below was diluted 1:10 before loading into the hemocytometer What is the concentration of the original culture? (Use appropriate notation and units) Was the hemocytometer loaded properly? How can you tell? a. b. Atter performing the hemocytometer count, you have exactly 10 ml of culture You require enough cells to perform your experiment? e exactly 1 x 106 cells to perform your experiment. Do you have C. 10. What is the template for your cDNA synthesis reaction? 11. What is the total volume of one cDNA synthesis reaction? 12. What is the purpose of RNase H, and when is it used? 13. What is the difference between First-Strand Synthesis using Random Primers and First Strand Synthesis using Oligo (dT)s? 14. Why are Random Primers a better choice for cDNA synthesis of a potentially degraded template? 15. You want to use 2 g of total RNA for your cDNA reactions. If your RNA concentration is 100 Jul, what volume of RNA will you add to your reaction? What volume of water? 16. Why is it necessary to convert RNA to cDNA before performing PCR? 17. What happens next week to the samples that you will prepare today? 18. Why do we add Ethanol to the spin column if it is so hard to remove?Explanation / Answer
ANS 1) Multigene households throw a specific twist into the problem of polymerase chain reaction (pcr) primer layout. one often needs primers as a way to no longer most effective expand the sequence of interest, however on the same time fail to extend the set of very similar sequences that contains the ultimate individuals of the circle of relatives. in a “regular” pcr experiment, one have to fear about “random” priming on unrelated sequences. with multigene households, one should try and keep away from nonrandom priming on related sequences with the aid of exploiting differences that distinguish the sequence of hobby. most “primer layout” packages do now not assist with this hassle. right here we speak what is involved in designing subfamily-specific primers.
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ANS 2) Autophagy is the natural, regulated, detrimental mechanism of the mobile that disassembles pointless or dysfunctional components. it also allows the orderly degradation and recycling of mobile additives.[4][5] in macroautophagy, centered cytoplasmic constituents are isolated from the relaxation of the cellular within a double-membraned vesicle referred to as an autophagosome.the autophagosome sooner or later fuses with lysosomes and the contents are degraded and recycled. three forms of autophagy are usually defined: macroautophagy, microautophagy, and chaperone-mediated autophagy (cma). in disorder, autophagy has been seen as an adaptive reaction to strain, which promotes survival, while in different instances it appears to sell cellular demise and morbidity. in the extreme case of starvation, the breakdown of cellular components promotes mobile survival by preserving cellular electricity ranges.
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ANS 3) A microbial populace is inoculated into a fresh medium, growth usually does no longer start immediately however only after a period of time referred to as the lag segment, which may be quick or prolonged relying at the history of subculture and increase situations.
condition of bacterial lifestyle used:
1)exponentially developing tradition inoculated into same medium: no lag section.
2)old or stationary culture inoculated into equal medium: lag phase (time required for bacteria to synthesize important cell constituent and if you want to go for binary fission).
3)broken bacterial way of life (warmness, radiation or toxic chemical substances): long lag section (time required to repair harm cells and time required to synthesize mobile parts)
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ANS 4) Starving can synchronize the cellular cycle for certain but how long need to you starve the cells will be a sensible trouble. i paintings on pulmonary artery endothelial cellular, primary mobile lifestyle, and observed out the tolerance to serum starve is pretty variable. i wager a498 cellular lines maybe greater proof against serum starve considering that it's far a cancer mobile line. to serum starve for 18 hours (in a single day) may also give you a better result. i recommend you to mix your compounds, and the control chemical or solvent, into the brand new way of life medium (may be 2-10% fbs) then pour onto the cells. the hassle about while to measure the proliferation will depend upon which assay you plan to use (mtt, xtt, brdu, or absolutely cellular be counted).
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ANS 5) An autophagosome is a spherical structure with double layer membranes. It is the key structure in macroautophagy, the intracellular degradation system for cytoplasmic contents (e.g., abnormal intracellular proteins, excess or damaged organelles) and also for invading microorganisms.the function of the tor signaling pathway and autophagy in regulating circadian rhythms primarily based on the behavior and structural plasticity of the lamina l2 monopolar cellular dendritic bushes. in addition, we tested the cyclic expression of the tor signaling pathway (tor, pi3k elegance 1, akt1) and autophagy (atg5 and atg7) genes in the fly’s brain. we found that tor, atg5 and atg7 showcase rhythmic expressions in the brain of untamed-type flies in day/night conditions (ld 12:12) which can be abolished in per01 clock mutants. the silencing of tor in in line with expressing cells shortens a length of the locomotor activity rhythm of flies. similarly, silencing of the tor and atg5 genes in l2 cells disrupts the circadian plasticity of the l2 cellular dendritic timber measured in the distal lamina. in turn, silencing of the atg7 gene in l2 cells adjustments the pattern of this rhythm. our effects indicate that the tor signaling pathway and autophagy are concerned inside the regulation of circadian rhythms inside the conduct and plasticity of neurons inside the mind of adult flies.
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