Minipreps (alkaline lysis plasmid purification) behind the scenes скачать в хорошем качестве

Minipreps (alkaline lysis plasmid purification) behind the scenes

Aka ALKALINE LYSIS, “minipreps” is a technique in which we separate and purify the plasmid DNA we put into bacteria (the DNA we want) from all the stuff that was already in the bacteria (the DNA we don’t want, proteins, sugars, etc.). You can’t just break the cells open (lyse them) & pull out all the DNA because the bacteria has its own DNA which you aren’t interested in. But some cool chemistry comes to the rescue!     longer text & figures in blog: http://bit.ly/minipreps   So we want to purify our plasmid DNA (pDNA), but that plasmid is in bacterial cells which have their own genomic DNA (gDNA) as well as a bunch of proteins and stuff. So how do we isolate the pDNA?    Clearly, we’ll need to get it out of the cells, which we can do by breaking the cells open (LYSIS). But before you break the cells open, you want to make sure they have an ideal environment to come out into. The cell membrane provides a nice natural barrier to allow you to swap out the external environment - so take advantage of it!    Right now, the cells are in the liquid media you grew them in (often Lysogeny Broth, LB) – in addition to the antibiotic for selection, the media contains salts, peptides (from the tryptone - trypsin-digested casein protein), vitamins etc. (from the yeast extract), all sorts of stuff the bacteria want/need to survive and thrive (and make lots of copies of your plasmid). http://bit.ly/bacterialmedia     If our goal is to purify the pDNA, we might as well get rid of as much of that stuff we don’t want now, while the cell membrane provides a convenient natural barrier (especially because some of that stuff could interfere with future steps). We also want to minimize the volume we’re working with. Right now, the cells are “spread out” in excess media but if we “spin them down” by centrifugation, we can separate the cells (heavy) from the media (light).    So we stick the tubes in a centrifuge, which spins really fast, pelleting the cells. We then pour off the liquid (SUPERNATANT) but keep the pellet which has our cells.     Now we resuspend them in a smaller volume of a more ideal solution. We’re going to be using a series of different solutions we refer to as “buffers.” “Buffer” is in reference to their pH-stabilizing ability which comes from component(s) that can both give protons (act as an acid) and take protons (act as a base). But we often use the term “buffer” pretty broadly to refer to different pH-stabilized “salt waters” http://bit.ly/phbuffers     The first buffer we’ll use in the miniprep is RESUSPENSION BUFFER (“P1” in this QIAprep kit (this isn’t a paid endorsement or anything, just what our lab uses) has:    ∙ Tris – this acts as the actual pH buffer (pH stabilizer) – it’s here because we’re not ready to change the pH yet    ∙ glycerol – this is an osmotic balancer – osmosis refers to the movement of water from where water is more concentrated (there’s less other stuff around) to where water is less concentrated (there’s more other stuff in between the water molecules). Since there’s a lot of stuff in the cells, if there isn’t a lot of stuff outside the cells, water will flood in. So we add glycerol to increase the amount of stuff outside the cells and thus keep the cells from bursting before we’re ready. Later we’ll burst them with detergent (synthetic soap), not pressure differences    ∙ RNaseA – this degrades RNA (and is why P1 has to be stored in the fridge) – we don’t want the RNA, so might as well break it down so it doesn’t clog things up or try to tag along (a lot of times in the lab we’re trying to protect from RNaseA, because this RNA chewer is all over the place (for example, bacteria often secrete it to destroy foreign RNA (like from viruses) before they can invade), but here our usual enemy is our friend because we’re both on team DNA! (RNaseA won’t chew DNA because it works in a sneaky way that gets the RNA to attack itself using its 2’ oxygen (which DNA doesn’t have - hence the d in DeoxyriboNucleic Acid) https://bit.ly/rnaseadepc     note: with this kit you have to add the RNaseA to the buffer (and then check the check box on top of the bottle to signal you did)    ∙ EDTA – this is a chelating agent (metal-biter) - it binds divalent cations (molecules with a +2 charge) like Mg²⁺ or Ca²⁺ which DNases (DNA chewers) need – prevents DNases from degrading the plasmid (RNaseA doesn’t need cation cofactors so it can still work and chew away the RNA).     finished in comments