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The stacking gel concentrates proteins loaded into the sample wells so that they are resolved as a unified "line" once they enter the stacking gel. The reason for the lower pH is that this "lower ionic strength implies higher electrical resistance and consequently a higher electric field, provoking the faster movement of the proteins and of every other ...


The other major difference between the two is the amount of acrylamide in the upper (stacking) gel - it's generally around 4%, while the lower (resolving) gel can vary from 6 or 8% to 20%, depending on the size of the protein(s) you're looking for. When you load your samples in the wells at the top of the gel, then start the current, not all of the sample ...


As many kits suggest, RNA concentration has the most profound influence on precipitation efficiency / recovery fraction. Time, temperature and precipitation agent concentration has lesser effect. See for example instruction by Life Technologies on LiCl precipitation: The Use of LiCl Precipitation for RNA Purification


As far as I know this has never been thoroughly analyzed for RNA, but there is an excellent paper on the precipitation of DNA and the usual conditions in BRL Focus by Zeugin (see below for the paper). Since DNA and RNA are pretty much the same (except for one OH-Group) and the conditions used for precipitation are also similar, I think we can use this for a ...


I think it is relatively unlikely that the RNA will degrade under this conditions. For the future, I would handle this differently: You can centrifuge the cells and snap freeze them in an appropriate buffer in liquid nitrogen and then store them at -80°C. I would not freeze the dry pellet, as these are often hard to re-dissolve after freezing. ...


PBS is isotonic to most cells, so in this buffer your bacterial cells are intact. Centrifuging them to remove PBS won't harm them it is quite standard procedure. You have to be careful after adding 2% SDS because it will lyse your cells and then the RNA contained within them will become "free" and RNAs are fragile molecules.


I'm assuming that you have a solution of bacteria in your buffer directly after growing them for some time, and without performing a lysis step yet. The 2% SDS step looks like it is for lysis of your cells. In that case, centrifuging the bacteria and resuspending them in lysis buffer is a very common step in many protocols. Separating whole bacteria from ...

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