第三讲NDA的分离与纯化.pptVIP

Sudden appearance of “white” precipitate after centrifugation; possible bi-products of 20 % SDSCrystallized insoluble white precipitate located on bottom of ependorf tube; signs of contaminants * This experiment fits well into exploring cell structures: Where is the DNA located? What properties of cell membranes allow us to break them open with detergents? Why is protease used to assist in extraction of DNA? Further discussion later in workshop. * Examples of cell types * Graphic showing Na+ ions associating with negatively charged phosphate groups on DNA backbone. * If anyone ends up with a lot more than 5 mls (7 mls or more), they need to remove the excess and discard it. DNA precipitation may not occur optimally if much more than 5 mls of cell lysates are present when alcohol is added during the precipitation step. It’s important NOT to shake the lysed cells – this will shear the DNA (break it into small fragments) and shorn DNA will not precipitate well. * * It is important to add the alcohol gently and keep tube at a 45 degree angle. After a few minutes, they should see small bubbles with DNA strands attached. If they see a nice clump of DNA, they can remove it to necklace vial before inversion. Alternatively, they can gently mix/tilt their test tube to see the DNA come out of solution and then make a necklace. Its important NOT to shake and dissociate the DNA that is precipitating. * First, remove the plastic stopper from the vial (you will replace the stopper after transferring your DNA). Don’t lose the stopper! It’s small and hard to see. Next, using a disposable transfer pipet, carefully transfer the DNA strands to the glass amulet. Don’t overfill the amulet; the stopper will be difficult to seal on the vial if there is too much liquid inside. * Can talk about lipid bilayer model of cell membranes and how detergents work. * Histones, which cause DNA to supercoil (compact) in the chromosome, are proteins. * Histones, which cause DNA to supercoil (com