281 Matching Annotations
  1. Last 7 days
    1. Bloomington Drosophila Stock Center

      DOI: 10.1038/s41598-023-46616-9

      Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

      Curator: @maulamb

      SciCrunch record: RRID:SCR_006457


      What is this?

    2. pucE69

      DOI: 10.1038/s41598-023-46616-9

      Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

      Curator: @maulamb

      SciCrunch record: RRID:SCR_006457


      What is this?

    3. da-GAL4

      DOI: 10.1038/s41598-023-46616-9

      Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

      Curator: @maulamb

      SciCrunch record: RRID:SCR_006457


      What is this?

    4. w1118

      DOI: 10.1038/s41598-023-46616-9

      Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

      Curator: @maulamb

      SciCrunch record: RRID:SCR_006457


      What is this?

  2. May 2024
    1. #32198

      DOI: 10.1186/s13064-023-00177-9

      Resource: (BDSC Cat# 32198,RRID:BDSC_32198)

      Curator: @maulamb

      SciCrunch record: RRID:BDSC_32198


      What is this?

    2. #34977

      DOI: 10.1186/s13064-023-00177-9

      Resource: (BDSC Cat# 34977,RRID:BDSC_34977)

      Curator: @maulamb

      SciCrunch record: RRID:BDSC_34977


      What is this?

    3. Bloomington Drosophila Stock Center

      DOI: 10.1186/s13064-023-00177-9

      Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

      Curator: @maulamb

      SciCrunch record: RRID:SCR_006457


      What is this?

  3. Apr 2024
    1. Article 122-9Version en vigueur depuis le 01 septembre 2022Modifié par LOI n°2022-401 du 21 mars 2022 - art. 6N'est pas pénalement responsable la personne qui porte atteinte à un secret protégé par la loi, dès lors que cette divulgation est nécessaire et proportionnée à la sauvegarde des intérêts en cause, qu'elle intervient dans le respect des conditions de signalement définies par la loi et que la personne répond aux critères de définition du lanceur d'alerte prévus à l'article 6 de la loi n° 2016-1691 du 9 décembre 2016 relative à la transparence, à la lutte contre la corruption et à la modernisation de la vie économique. N'est pas non plus pénalement responsable le lanceur d'alerte qui soustrait, détourne ou recèle les documents ou tout autre support contenant les informations dont il a eu connaissance de manière licite et qu'il signale ou divulgue dans les conditions mentionnées au premier alinéa du présent article. Le présent article est également applicable au complice de ces infractions. Conformément à l’article 18 de la loi n° 2022-401 du 21 mars 2022, ces dispositions entrent en vigueur le premier jour du sixième mois suivant sa promulgation.
    1. Article 122-9Modifié par LOI n°2022-401 du 21 mars 2022 - art. 6N'est pas pénalement responsable la personne qui porte atteinte à un secret protégé par la loi, dès lors que cette divulgation est nécessaire et proportionnée à la sauvegarde des intérêts en cause, qu'elle intervient dans le respect des conditions de signalement définies par la loi et que la personne répond aux critères de définition du lanceur d'alerte prévus à l'article 6 de la loi n° 2016-1691 du 9 décembre 2016 relative à la transparence, à la lutte contre la corruption et à la modernisation de la vie économique. N'est pas non plus pénalement responsable le lanceur d'alerte qui soustrait, détourne ou recèle les documents ou tout autre support contenant les informations dont il a eu connaissance de manière licite et qu'il signale ou divulgue dans les conditions mentionnées au premier alinéa du présent article. Le présent article est également applicable au complice de ces infractions. Conformément à l’article 18 de la loi n° 2022-401 du 21 mars 2022, ces dispositions entrent en vigueur le premier jour du sixième mois suivant sa promulgation.
  4. Feb 2024
    1. Résumé de la vidéo [00:00:00][^1^][1] - [00:16:42][^2^][2]:

      Cette vidéo est un webinaire présenté par Serge, un représentant syndical du SNAC, qui explique la Dotation Horaire Globale (DHG) dans les établissements scolaires français. Il détaille comment la DHG est calculée, distribuée et utilisée pour organiser les emplois du temps, ainsi que l'importance du Tableau de Répartition des Moyens (TRM) dans la gestion des ressources et la prise de décisions pédagogiques au sein des établissements.

      Points clés: + [00:00:17][^3^][3] Qu'est-ce que la DHG * Définition et rôle + [00:01:03][^4^][4] Calcul de la DHG * Facteurs influençant la DHG + [00:02:04][^5^][5] Temporalité de la DHG * Processus et timing + [00:03:01][^6^][6] Mythes sur la DHG * Clarification des idées reçues + [00:04:01][^7^][7] Importance du TRM * Répartition et impact sur l'établissement + [00:06:07][^8^][8] Processus de décision du TRM * Étapes et implications + [00:08:00][^9^][9] Si le TRM est refusé * Procédures et alternatives + [00:10:36][^10^][10] Responsabilités en cas de refus du TRM * Actions du chef d'établissement + [00:13:01][^11^][11] Conseils pour l'administration * Importance de la participation et de la proposition

  5. Jan 2024
  6. Nov 2023
    1. 3. qu’est-ce que La promotioninterdite d’entreprisecommerciaLe ?Evidemment, l’article D.111-9 du code de l’éducationimplique que les associations de parents d’élèves sevoient interdire la diffusion de « publicités » au senscourant
  7. Oct 2023
    1. a small dog.

      Thread.1

    2. Poe's source for his detective is the philosophic protagonist of Voltaire's Zadig

      Poe's source of inspiration

    3. I do not mean to say they are not ingenious — but people think they are more ingenious than they are — on account of their method and air of method

      Characteristic of the reader in writer's mind

    4. It is the ancestor of a vast number of works which have given much harmless pleasure to all sorts and conditions of men

      It has a profound effect

  8. Sep 2023
    1. “If now, in addition to all these things, you have properly reflected upon the odd disorder of the chamber, we have gone so far as to combine the ideas of {dd}an agility astounding, a strength superhuman,{dd} a ferocity brutal, a butchery without motive, a grotesquerie{e} in horror absolutely alien from humanity, and a voice foreign in tone to the ears of men{f} of many nations, and devoid of all distinct or intelligible syllabification. What result, then, has ensued? What impression have I made upon your fancy?”

      I love how Dupin uses "Fancy" at the end of the sentence. Since it emphasize again the case is not normal, so you have to fancy it and see it with your imagination.

    2. In a word, why did he abandon four thousand francs in gold to encumber himself with a bundle of linen? The gold was abandoned. Nearly the whole sum mentioned by Monsieur Mignaud, the banker, was discovered, in bags, upon the floor. I wish you, therefore, to discard from your thoughts the blundering idea of motive{m} engendered in the brains of the police by that portion of the evidence which speaks of money delivered at the door of the house.

      The motivation is not out of money but something peculiar.

    3. This relieves us of all doubt upon [page 549:] the question whether the old lady{c} could have first destroyed the daughter, and afterward{d} have committed suicide. I speak of this point chiefly for the sake of method; for the strength of Madame L’Espanaye would have been utterly unequal to the task of thrusting her daughter's corpse up the chimney as it was found; and the nature of the wounds upon her own person entirely preclude the idea of self-destruction. Murder, then, has been committed by some third party; and the voices of this third party were those heard in contention. Let me now advert — not to the whole testimony respecting these voices — but to what was peculiar{e} in that testimony. Did you observe any thing peculiar about it?” I remarked that, while all the witnesses agreed in supposing the gruff voice to be that of a Frenchman, there was much disagreement in regard to the shrill, or, as one individual termed it, the harsh voice.

      They know that the testimonies might already lead the observation in the wrong way, which is earlier to be accepted. However, it limits the imagination of thinking out of the box.

    4. There was something in his manner of emphasizing the word “peculiar,” which caused me to shudder, without knowing why. “No, nothing peculiar,” I said; “nothing more, at least, than we both saw stated in the paper.”

      We see that the attitudes of the narrator and Dupin toward the case are so different. And the narrator's perspective is more like usual but this case is obviously strange and peculiar as it is mentioned. Therefore, this two sentences highlight again and alert the reader - the case should be thought the other sad around.

    5. Upon reaching the first landing, heard two voices in loud and angry contention — the one a gruff voice, the other much shriller — a very strange voice. Could distinguish some words of the former, which was that of a Frenchman. Was positive that it was not a woman's voice. Could distinguish the words ‘sacré’{s} and ‘diable.’ The shrill voice was that of a foreigner. Could not be sure whether it was the voice of a man or of a woman. Could not make out what was said, but believed the language to be Spanish.{t}

      This is the first testimony mentioned the voices of the murderer, which to the point of the core characteristics of the murderer. Thus, the readers might be easily misled that the murderer is human.

    6. Did not see any person in the street at the time. It is a bye-street{f} — very lonely.

      From this testimony, we can see that it implies that no other people know the old lady had huge amount of money at home.

    7. “Several witnesses, recalled, here testified that the chimneys of all the rooms on the fourth story were too narrow to admit the passage of a human being.

      This is the first hint to ask reader think out of the box, maybe the murderer is not a regular murderer, maybe the murderer is not even a human being.

    8. You will say that it might have been the voice of an Asiatic — of an African. Neither Asiatics nor Africans abound in Paris; but, without{l} denying the inference, I will{m} now merely call your attention to three points.{n} The voice is termed by one witness ‘harsh rather than shrill.’ It is represented by two others to have been ‘quick and unequal.’ No words — no sounds{o} resembling words — were{p} by any witness mentioned as distinguishable.

      From this point, we can see that Dupin see this case from the other side. He did not take all the testimonies as the proof or observation but try to link them together to see the whole picture of the murderer.

    9. whisker

      Whisker is any of the long, stiff hairs growing on the face of a cat, mouse, or other mammal.

      鬍鬚

    10. agility

      Agility is the ability to move your body quickly and easily.

      靈敏

    11. Jardin des Plantes

      Jardin des Plantes is French for "Garden of the Plants."

    1. “I am now awaiting,” continued he, looking toward{z} the door of our apartment — “I am now awaiting a person who, although perhaps not the perpetrator of these butcheries, must have been{a} in some measure implicated in their perpetration. Of the worst portion of the crimes committed, it is probable that he is innocent.

      Dupin is speculating about who the person he might be and arguing that he is innocent. Dupin is right.

    2. “The Purloined Letter,”

      It is another detective short story written by Allan Poe.

    3. I do not mean to say they are not ingenious — but people think they are more ingenious than they are — on account of their method and air of method

      The author was slightly annoyed at the attention which was paid to the Dupin stories.

    1. Because adding research to your essays requires the use of others’ writing, and because many assignment require you to write about others’ writing, it is possible to plagiarize accidentally through incorrect citation.

      When we’re writing an essay, we often quote others writing in our paper, that might cause some copyright issues.

    2. Using ideas from elsewhere requires correct citation, and using ideas from elsewhere without citation is plagiarism.

      If you need to use ideas from elsewhere, you must require correct citations. Don’t use other’s creation!

  9. Jun 2023
    1. kons-9 is that it combines the power of a software development IDE with the visual tools of 3D graphics authoring system. It does this by being implemented in Common Lisp, an object-oriented dynamic language which provides powerful facilities for exploratory development and rapid prototyping within a live interactive software environment

      IDE + 3D + Lisp = Unique features:: * software development IDE with visual toold of 3d graphics authoring system * unlimited extensibility (no distinction between developers and end users)

      https://youtu.be/NJe4isZ7NHI

    2. REPL-based

      Read-Eval-Print Loop and is a software programming environment that allows developers to interactively execute their code

  10. Apr 2023
    1. I figure that most of the learning students lost in Zoom school is learning they would have lost by early adulthood even if schools had remained open.

      Here we have a correlation between zoom school and the feeling of missing out on the school experience. Is this true? Well he states "I figure" so this doesn't typically mean there is evidence backing up the claim and there also is no article linked to this part of the essay. One way to do this is to survey children in Zoom school and collect data from the numbers.

    1. Most grown-ups believe that school is only for learning back-to-back. They don’t understand the parts that build character for us teens.

      Grown ups, sometimes, don't understand what school is like now compared to when they were in school. What I disagree with is the fact that grown ups don't understand that it builds character. That is one thing that hasn't changed over the years. Making life long friends and finding yourself. The statement leaves out these important factors. We have to consider the statement from every angle.

    1. 2.7-9 Theorem (Continuity and boundedness)

      Let T be a linear mapping between 2 normed space, then:

      1. T is continous if and only if it's bounded.
      2. T is continous at a single point then it's continous.
    2. 2.6-9 Theorem (Range and null space).
      1. Range is a vector space.
      2. If the dim of the range is less than infinitely, then the dim of the range is \(\le\) dim of the domain.

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  11. Feb 2023
    1. rocessing of special categories of personal data 45. Subject to compliance with the Data Protection Regulation and any other relevant enactment or rule of law, the processing of special categories of personal data shall be lawful to the extent the processing is— (a) authorised by section 41 and sections 46 to 54 , or (b) otherwise authorised by Article 9.

      scd #specialcategoriesdata

    1. collecting and checking the content of declarations of private interests, of personal data that are liable to disclose indirectly the political opinions, trade union membership or sexual orientation of a natural person constitutes processing of special categories of personal data, for the purpose of those provisions.

      Second question: If you collect it, can you infer from it?

    2. those provisions cannot be interpreted as meaning that the processing of personal data that are liable indirectly to reveal sensitive information concerning a natural person is excluded from the strengthened protection regime prescribed by those provisions, if the effectiveness of that regime and the protection of the fundamental rights and freedoms of natural persons that it is intended to ensure are not to be compromised.

      And here's the key element for indirect/inferred data. In order for Article 9 to matter, it must also include data that infers SCD.

  12. Jan 2023
    1. surveillance capitalism, invented by Google in 2001, benefitted from a couple of important historical windfalls. One is that it arose in the era of a neoliberal consensus around the superiority of self-regulating companies and markets. State-imposed regulation was considered a drag on free enterprise. A second historical windfall is that surveillance capitalism was invented in 2001, the year of 9/11. In the days leading up to that tragedy, there were new legislative initiatives being discussed in Congress around privacy, some of which might well have outlawed practices that became routine operations of surveillance capitalism. Just hours after the World Trade Center towers were hit, the conversation in Washington changed from a concern about privacy to a preoccupation with “total information awareness.” In this new environment, the intelligence agencies and other powerful forces in Washington and other Western governments were more disposed to incubate and nurture the surveillance capabilities coming out of the commercial sector.

      !- summary : surveillance capitalism incentives - popularity of neoliberal norm of minimizing government regulation - 9/11 accelerated popularity of a global surveillance state

  13. Dec 2022
  14. Oct 2022
    1. For the second time Goutor mentions using different size cards for different note types, but doesn't specifically advise for it or provide a reason. Perhaps his advice for consistency and card size applies only to cards of particular types? (p28)

      link to: https://hypothes.is/a/XPphjkNZEe2s3i9VV4qt1g


      Incidentally he also specifically mentions 7x9" cards here too. How frequently used were these as a standard?

  15. Aug 2022
  16. Jun 2022
    1. User participation in any online internet community generally follows the 90-9-1 rule:90% of community members are lurkers who read or observe, but don’t contribute9% of community members edit or respond to content but don’t create content of their own1% of community members create new content
  17. Mar 2022
    1. in which we retaliated for an attack on our soil

      Retaliation should have been directed against wahabis in S>Arabia - occupying a whole nation to hunt down a person is a sentimental but flimsy pretext.

  18. Dec 2021
    1. These designers value expression over style

      It's about expressing oneself or creates distortion rather than valuing or achieving a certain look that they should convey.

    2. syntactical

      in a way that relates to the grammatical arrangement of words

      in a sentence: a syntactically complicated language

      definition

    3. Shattering the constraints of minimalism was exhilarating and far more fun than the antiseptic discipline of the classical Swiss school.

      That's understandable because doing the same type of style over and over again can be boring sometimes.

  19. Nov 2021
    1. As a result, the Texas law is a permissible regulation of speech.

      incorrect, they ruled that Johnson's actions did not threaten to disturb the peace or that the state's interests in preserving the flag as a symbol.... justify his criminal conviction for engaging in political expression

    1. Only certain religious groups are free to participate.

      I don't think this is apart of the opinion, I cant find where it says that only certain groups are free to participate.

  20. Oct 2021
  21. Sep 2021
    1. Happiness

      Rejoice, O young man, in your youth, and let your heart cheer you in the days of your youth. Walk in the ways of your heart and the sight of your eyes. But know that for all these things God will bring you into judgment.

  22. Jun 2021
    1. For example, saver's credits make certain retirement savings tax-exempt, meaning savers get to keep more of the interest they earn on savings investments.

      This example is pulled from Giacomo's slides. Possible replacement: education fund accounts? (Same concept of tax-exemption, less common)

    2. In the United States and many other countries, the government taxes gains from private investment. Low capital gains taxes encourage investment and so also economic growth.

      This sentence is from OS

  23. May 2021
  24. Mar 2021
    1. Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

      AssayResult: 5.2

      AssayResultAssertion: Normal

      StandardErrorMean: 0.39

    2. A total of 84 PALB2 patient-derived missense variants reported in ClinVar, COSMIC, and the PALB2 LOVD database were selected

      HGVS: NM_024675.3:c.1189A>T p.(Thr397Ser)

    1. SUPPLEMENTARY DATA

      AssayResult: 92.68

      AssayResultAssertion: Not reported

      PValue: > 0.9999

      Comment: Exact values reported in Table S3.

    2. To this end, 44 missense variants found in breast cancer patients were identified in the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar) and/or selected by literature curation based on their frequency of description or amino acid substitution position in the protein (Supplemental Table S1).

      HGVS: NM_024675.3:c.242A>G p.(Lys81Arg)

    1. Source Data

      AssayResult: 95.74

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardErrorMean: 14.87

      Comment: Exact values reported in “Source Data” file.

    2. Source Data

      AssayResult: 62.31

      AssayResultAssertion: Not reported

      ReplicateCount: 2

      StandardDeviation: 11.49

      StandardErrorMean: 8.13

      Comment: Exact values reported in “Source Data” file.

    3. We, therefore, analyzed the effect of 48 PALB2 VUS (Fig. 2a, blue) and one synthetic missense variant (p.A1025R) (Fig. 2a, purple)29 on PALB2 function in HR.

      HGVS: NM_024675.3:c.13C>T p.(P5S)

    1. Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function

      AssayResult: 32

      AssayResultAssertion: Abnormal

      ReplicateCount: 31

      StandardErrorMean: 5

      Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

    2. we selected 73 previously unstudied variants: 63 suspected Brugada syndrome variants and 10 suspected benign variants

      HGVS: NM_198056.2:c.1186G>C p.(Val396Leu)

  25. Feb 2021
    1. He was so deeply offended and concerned by the notion that somehow it was America’s fault that a group of radical, violent Islamist terrorists killed nearly 3,000 Americans that it opened his eyes to the indoctrination that was happening in his classes.

      This seems to be true revisionist history. No one I've come across took a "blame America first" approach. Gingrich and Miller would be incredibly hard pressed to come up with contemporaneous statements that back up this proposition.

    1. Supplemental material

      AssayResult: 45

      AssayResultAssertion: Normal

      Comment: See Table S2 for details

    2. Supplemental material

      AssayResult: 5.4

      AssayResultAssertion: Abnormal

      Comment: See Table S2 for details

    3. We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.

      HGVS: NM_000546.5:c.216dup p.(Val73Argfs*76)

  26. Oct 2020
    1. Figure 9 shows the decreasing optical transmit-tance at the visible region with increasingfilm thickness(increasing immersion time)

      La figura 9 muestra el decrecimiento de las transmitancia óptica en la región visible con un incremento del grosor de la película (incrementando el tiempo de inmersión).

    Tags

    Annotators

    1. Men who worship me,thinking solely of me,always disciplined,win the reward I secure.

      it state in this part that the man who worship me and think about me will be reward at the end by lord.

    2. I am the universal father,mother, granter of all, grandfather,object of knowledge, purifier,holy syllable OM, threefold sacred lore.

      Does Krishna showing his power to the world by calling himself as a universal father?

  27. Sep 2020
    1. 71 1. The Standard Version, Tablet IX [He] clad himself in their skins, he ate their flesh. Gilgamesh [dug] wells that never existed before, [he] drank the water, as he chased the winds. Sham ash grew worried, and bending down, he spoke to Gilgamesh: '0 Gilgamesh, where are you wandering? The life that you seek you never will find.' Said Gilgamesh to him, to the hero Shamash: 'After roaming, wandering all through the wild, when I enter the Netherworld will rest be scarce? I shall lie there sleeping all down the years! 'Let my eyes see the sun and be sated with light! The darkness is hidden, how much light is there left? When may the dead see the rays of the sun?'

      Gilgamesh seemed sad after the death of his friend Enkidu.

    2. The life that you seek you never will find.'

      Shamash was worried about Gilgamesh the death of Enkidu.

  28. Jul 2020
  29. Jun 2020
  30. Mar 2020
  31. Nov 2019
    1. Christian Reclamation Center

      christain reclamation center potential workplace

    2. a drive-in restaurant

      drive in near funeral home, reached by car

    3. THE BUDDHIST church up on the hill next to a playground

      buddhist church were mama's funeral was held

    1. Überwachung

      Abseits vom Kapital, welches aus solcher Überwachung geschlagen wird, erscheint mir die Überwachung an sich als ebenso interessant. Beispielsweise die staatliche Überwachung Aller aus Gründen der "inneren Sicherheit". Diese wir am Beispiel der Folgen von 9/11 und der Snowden-Affäre von eben diesem in der 1368. Folge des Podcasts "The Joe Rogan Experience" im Detail aufgeschlüsselt.

    1. Überwachungs

      Abseits vom Kapital, was aus solcher Überwachung geschlagen wird, erscheint mir die Überwachung an sich als ebenso interessant. Beispielsweise die staatliche Überwachung Aller aus Gründen der "inneren Sicherheit". Diese wird am Beispiel der Folgen von 9/11 und der Snowden-Affäre von eben diesem in der 1368. Folge des Podcasts The Joe Rogan Experience im Detail aufgeschlüsselt.

  32. Jul 2019
    1. Analysis of internal transcribed spacer region
    2. Effect of temperature on xylanase activity
    3. Effect of pH on xylanase activity
    4. Effect of various carbon sourceson xylanase activity
    5. Assayof xylanase activity
    6. Dinitrosalicylate reagent (DNS)(per liter)
    7. Citrate phosphate buffer
    8. Reagents
    9. Xylanase activity
    10. Harvesting of cultures
    11. Enzyme production (EP) medium
    12. Inoculum preparation
    13. Sporulation medium used for Trichodermasp
    14. Maintenance of Trichodermasp. culture
    15. Sterilization
    16. Materials for xylanase induction
    17. Induction of xylanase from Trichodermasp
  33. Jun 2019
    1. Purified HbS was digested with carboxypeptidase B (200 mg hemoglobin to 1 mg of the enzyme) for 3 hours in freshly prepared 0.05 M Tris-acetate buffer pH 7.1) at 25°C , followed by passage through a cation-exchange column using Whatman CM52.
    2. Preparation of HbS{des arg 141a
  34. May 2019
    1. On December 14, 2017, FCC commissioners revoked Network Neutrality rules by a 3–2 vote. As a result, ISPs can now legally offer “tiered service” favoring some websites, services, and applications with faster connections, blocking others, or charging some conNet Neutrality Why It Matters to School Librarians Feature ARTICLE tent providers greater fees to connect to their customers (Fung, 2017). This is the “fast lane” and “slow lane” concept

    1. Protein concentrations were detennined using BCA protein estimation kit (Pierce, .. USA). The assay was perfonned according to the instructions provided by the manufacturer. Various dilutions of the sample or BSA were made in appropriate buffer and 200 J.ll of supplied reagent mix (1 :50 ratio) was added to each well in a 96 well plate. The plate was incubated at 37°C for 1 h and the absorbance was measured at 540 nrn.
    2. Protein Estimation
    1. antibody at an appropriate dilution. The immunoreactivity was detected by enhanced chemiluminescence using an ECL detection kit (Amersham Biosciences) and were recorded on X-ray films after appropriate exposure and development. It is important to note that the blots for probing phosphorylated proteins were performed using 1% BSA as blocking agent instead ofblotto
    2. Whole cell extracts were prepared by treating cells with lysis buffer (0.125M Tris, 4% SDS, 20% glycerol, and 10% ~-mercaptoethanol), and protein estimation was performed using CB-X protein assay kit as per manufacturer's protocol. Lysates were resolved on 12% SDS-PAGE gel, following which Western transfer was performed onto nitrocellular membranes using a BioRad Western transfer apparatus. The blots were incubated with 5% blotto (non-fat dry skimmed milk) in 0.05% PBS-Tween 20 for 1 h to block non-specific binding sites following which they were incubated for 1 h with primary antibody at an appropriate dilution prepared in 1% blotto in 0.05% PBS-Tween-20. The blots were washed 3x with 0.05% PBS-Tween-20 at 5 min intervals following which they were incubated for 1 h with secondary
    3. SDS-PAGE and Western blot
    1. Sequencing of cloned inserts was done by Sanger's dideoxy chain termination method (Sanger et al., J 977) using Sequenase version 2.0 kit from USB. 5 J.ll of J mg/ml suspension of plasmid DNA was incubated in denaturation buffer at 37 °C for 30 min. in a reaction volume of 50 J.ll, and then the precipitation was carried out in the presence of 5.5 J.ll of 3M sodium acetate and 4 volumes of chilled ethanol at -70 °C for 30 min. The pellet, obtained by centrifugation at 10,000 g, for 20 min., at 4 °C, was washed with 70% ethanol and resuspended in 7 J.ll of sterile water. J pmole of sequencing primer in J J.ll water and 2J.ll of 5X sequenase reaction buffer were added to denatured DNA and the reaction mix was incubated at 65 °C for 5 min. for primer annealing. The reaction mixture was cooled slowly to about 35 °C, by putting the heat block at room temperature. For labeling, J J.ll of 0. J M DTT, J J.ll radioactivity containinig J 0 J.lCi of 35S dATP, 2 Jlllabeling mix diluted 5-fold in steriJe water and 2 Jll sequenase enzyme diluted 8-fold in sequenase dilution buffer were added to the· primer annealed DNA. Incubated the reaction mixture at room temperature for 2-5 min. and added 3.5 J.ll to each of the 4 different tubes containing 2.5 J.ll dideoxy nucleotides ddATP, ddTTP, ddCTP, and ddGTP separately. The mixture was incubated at 37 °C for 5 min. and finally, the reaction was stopped by adding 4 J.ll of estop solution to each tube. Reaction products were separated on a 6% polyacrylamide sequencing gel made in TBE buffer containing 7.5 M urea. The samples were heated at 75 °C for 2 min. and immediately loaded on the gel. The gel was run at a constant power of 60 watts maintaining the temperature of gel between 50-55 °C, dried and exposed to an X-ray film.
    2. DNA Sequencing
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