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2

Addition to what Chris already said: Papain can be used for cells sensitive to trypsin (neurons etc) Collagenase can be used for certain cells where trypsin is ineffective (Accutase is a commercially available enzyme mix(?) which has collagenase activity) Hyaluronidase - I don't know where it is specifically preferred but it is used. Pronase and ...


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This addition to the protocol worked out and good RNA quality was obtained: Homogenization Homogenized the embryos, suspended (and frozen) in trizol by passing through 2ml insulin syringes. (About 10 times). Washing Washed 2 times with 500µl 80% Ethanol Washed 3 times with 500µl 70% Ethanol Dechorionation was dispensable and Proteinase-K treatment ...


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Non-homologous end joining indeed induces errors in the affected sequence. But you have to keep in mind, that NHEJ is an emergency repair mechanism which involves a "repair or die" chance. If the chromosomal break is not repaired it is not unlikely that the cell will get into apoptosis, or, even worse, develops into a cancer cell. Introducing small errors is ...


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NHEJ is indeed error prone. It is called "non-homologous" because it does not use a "homologous" template from another sequence-matching piece of DNA to guide the repair. Homologous repairs avoid causing mutations because the similar string of DNA acts as a template so that the cell knows what letters to put into the gap. When there's no template, there's ...


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Lets make this a proper answer: There are a few possibilities to detach adherent cells without Trypsin. PBS/EDTA: Integrins and Cadherins play an important role in the adhesion and also in maintaining cell-cell contacts. These function of these proteins depends on Calcium2+ ions, so EDTA will chelate them and make them unavailable. First remove the culture ...


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This is an important topic in immunology, especially for vaccine development. MHC or HLA is a molecule expressed by some cells of the immune system which acts like a "catcher's mitt" and "presents" short snippets of protein to other immune cells. Other molecules act alongside MHC to provide co-signals which promote or suppress immune attack against the ...


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DNA polymerase must catalyse the addition of 4 different nucleotides to the growing strand. This means that it cannot directly determine which base to incorporate at a specific point (how would it 'know' which base to incorporate and how it would it change its specificity for different bases). This means that the specificity for which base pair to ...


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High-fidelity DNA polymerases have several safeguards to protect against both making and propagating mistakes while copying DNA. Such enzymes have a significant binding preference for the correct versus the incorrect nucleoside triphosphate during polymerization. If an incorrect nucleotide does bind in the polymerase active site, incorporation is slowed ...


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There are two possible sources: One is the article from the Bethesda Research Laboratories (reference 1), which names a few, the other is the book "DNA cloning: A practical approach" (see reference 2)., which sits here in my bookshelf. Taking both together, the DH5 alpha strain is derived from the DH5 strain with the introduction of a few additional ...


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That is not true. If you had forgotten to add the antibiotic before inoculation then you can add it before the bacteria starts growing. Make sure you add it when the bacteria is still in lag phase. If you add it later then it won't be effective as some non-resistant (non-transformed) bacteria would have already expanded their population. Ampicillin and ...


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Welcome to BioSE! As @Chris mentioned, transported atoms and molecules don't typically retain a record of their past history, so just by looking at a molecule in the cell you can't in general tell how it arrived in the cell (an exception might be proteins that have some chemical modification on them that allows them to be imported via a specific mechanism, ...


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Proteinase K is sensitive for autolysis. The enzyme is stabilized by the presence of calcium ions (which bind to the protein). Absence of calcium promotes autolysis of the enzyme and also reduces the half life of the enzyme activity. The most likely reason why most researchers never notice an substancial effect of autolysis is that this takes around 48h to ...


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According to invitrogen proteinase K does undergo autolysis, but that some leftover fragments still have protease activity. The solution should be stored at -20C, this low temperature is probably the best way to prevent autolysis. If you're using the enzyme to digest protein at 37C, you'll just have to deal with losing the proteinase k during the reaction ...


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See this paper. They have studied RBP-protected sites in the entire human transcriptome by RNA-protein crosslinking followed by RNAse digestion and sequencing: PIPseq. Figure 1 of the paper shows distribution of protein protected sites in RNAs. They also correlate it with different regions of mRNA and its expression. They show number of protein protected ...


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The sRNA mediated regulation in bacteria operates via diverse mechanisms. This case of sRNA competing with a mRNA for a protein is a passive kind of regulation. This might be good for finetuning but may not be very efficient. It is much better to actively regulate a mRNA by direct binding. Also, it will work only if the concerned protein is in limiting ...


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PSI-BLAST is an iterative algorithm. Each cycle it uses a model (the position-specific scoring matrix, or PSSM) to search for sequences matching the model, next updates the model with the sequences found, and then runs the search again with the updated model. That P-value controls which of the sequences found in each iteration should be included in the new ...


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Anti-oxidants affect human metabolism by altering the redox states of the cell and redox-regulated functions and signaling mechanisms. The following quotes are from The Redox Stress Hypothesis of Aging (Free Radic Biol Med. Feb 2012) More recently, in a major conceptual shift, ROS have been found to be physiologically vital for signal transduction, ...


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Lots of interesting questions! Let me try to address a few of them as I don't think I am qualified to answer them all but hopefully I can get this thread started. I am a graduate student in the biophysical chemistry field and have been following a little bit of the Crispr Cas9 craze in the last couple of years. So I am not an expert on Cas9 by any means but ...


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The probability of evolution occurring is inconsequential. Suppose for instance, that their are a bunch of different outcomes for planets: a) Life b) a planet that consists of more than 90% gold c) A form of self replicating object that we can't recognize. etc... for an infinite amount of scenarios Let's suppose that each one of these scenarios has an ...



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