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TRIzol extractions of RNA, DNA, and protein скачать в хорошем качестве

TRIzol extractions of RNA, DNA, and protein 2 years ago

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TRIzol extractions of RNA, DNA, and protein
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TRIzol extractions of RNA, DNA, and protein

TRIzol - a hint at what it does is in the name, but no matter which of the 3 things it can help you extract that you want (RNA, DNA, or protein), the basic idea is the same… Take a solution containing a mix of molecules (often a cellular lysate - the cell guts you get when you break cells open) Add chemicals that take advantage of and accentuate differences between the different types of molecules Give the molecules a choice of solvents (things to dissolve in) and let them choose Centrifuge (spin really fast) to separate the different solvent portions (which will contain different molecules depending on which solvent those molecules chose) Take the solvent portion the molecule of interest chose to hang out with and add something to make it so that the molecule doesn’t want to hang out with that solvent any more (get it to precipitate out) Centrifuge to collect that precipitate into a pellet Remove the solvent Wash* the pellet with a solvent that will dissolve contaminants but not the molecule of interest *add wash solution and recentrifuge, then remove wash solution Add a solvent that the molecule really likes so that it will redissolve - now all alone! For the RNA you take the aqueous layer from that first extraction… Once you remove that, you’re left with the interphase & the organic phase. The DNA can be found in both the interphase and the organic phase so you want to work with them both as you move forward. For the DNA… You can get it to precipitate by adding EtOH. For the protein… You can get it to precipitate out of the liquid left over from the DNA precipitation. Just add isopropanol. Getting it to redissolve is the biggest challenge. It typically involves the use of the detergent SDS to help solubilize the clumps of protein. now here’s more detail on the RNA extraction part: blog form - text from May:  http://bit.ly/trizolRNAextraction ; YouTube:    • RNA extraction biochemistry (the TRIzol ak...     EXTRACTION works by separating molecules based on their SOLUBILITY (and insolubility) in different liquids, which we call solvents. It’s kinda like having a mixture get a “divorce” and letting the “kids” decide which parent’s house they want to live in after the split – in our case they can choose between an aqueous (water-based) “phase” or an organic (carbon-based), phenol-chloroform “phase.” We call those phases because they don’t mix with each other, so they will separate into layers (like oil and water).       And, when they separate, they’ll take molecules that are soluble in them with them. Being soluble means that each copy of a molecule (or a complex) has its own full coat of solvent. The individual dissolved molecules (solutes) don’t “fall apart,” they just “part” - molecules are made up of atoms (individual carbons, oxygens, nitrogens, etc.) connected via strong bonds called covalent bonds. These intRAmolecular (within a molecule) bonds don’t get broken when something dissolves (e.g. each sugar molecule stays a sugar molecule). Instead, only the weaker, noncovalent, intERmolecular (between molecule) attractions do, with solute-solute interactions being swapped for solute-solvent ones.       Most of the time in biochemistry, the solvent we’re talking about is WATER. Why? Our bodies are mostly water, so if we want to study what goes on in our bodies, we use water-based solutions. We call such water-based solutions AQUEOUS, which you will sometimes see abbreviated in equations with an italic subscript “aq.”.    It’s not just for the sake of “authenticity” that we study compounds in water. Because most molecules in our body have to work in a watery environment, they’ve evolved to function optimally in that environment, which involves being maximally soluble so that they can move around and do stuff.      If something’s NOT soluble, it means that the “don’t wannabe solute” molecules would rather bind to each other than to the solvent molecules (thing in which you’re dissolving) &/or the solvent molecules would rather bond to each other than to the solute.    If molecules bind to each other instead of the solute, they can clump up (aka aggregate) as precipitate. Doing this maximizes their contact with each other & minimizes their contact with the solvent, but it also makes them non-functional. You definitely don’t want this occurring in your cells, so you need the molecules to be soluble.     But there are solvents other than water, and different molecules like some solvents better than others, largely due to their charge distributions & if you give them options they can choose a different solvent instead of just panicking and clumping up.   finished in comments

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