General Question

ScottyMcGeester's avatar

What exactly does it mean to "resuspend the cell pellet"?

Asked by ScottyMcGeester (1030 points ) March 24th, 2014

I can’t go into much details about the experiment, but I’m not getting any results from trying to obtain DNA from E. coli cells.

I have E.coli cells in a pellet in a tube. The very first step is to resuspend it using this particular buffer. I know the problem I’m having has to be at the beginning because the setup is in such a way that I can tell where the issue must be if I get absolutely no results from controls or the test samples.

(If it isn’t the resuspension part, then my E.coli cultures must be bad, but I doubt it because they’re fresh and have a high absorbance)

When I do that resuspension step, I pipette the required amount into the tube. . .but the pellet doesn’t necessarily “suspend” in the liquid. It just stays there at the bottom. I invert the tube a little and part of the pellet breaks off. I’ve been told to always “be careful” with the pellet, so I’m confused as to what “resuspension” then should LOOK like when using it to resuspend a cell pellet. Do I have to break up the cell pellet?

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4 Answers

BhacSsylan's avatar

Yes, resuspension involves breaking up the cell pellet. It means to get the cells back into solution. Usually this involves vortexing the sample, which isn’t exactly gentle but at that stage of the procedure is usually not a problem. It’s only after lysis stocks are added that more care needs to be taken so that genomic DNA is not shredded.

ScottyMcGeester's avatar

@BhacSsylan Another situation now.

Say you have a DNA pellet. Not a cell pellet this time. Obviously it’s very small, but you assume it’s there in the tube after you wash it with ethanol. My procedure says “remove excess ethanol”. I’m unsure about how this is worded because it’s not exactly clear if that means “remove all the ethanol you used to wash the DNA” or “remove only some of it”.

I have gotten results before but the point is – what’s the best way to remove ethanol after washing a DNA pellet and then resuspending it? How am I sure that I didn’t accidentally pipette some DNA out when I removed the ethanol?

BhacSsylan's avatar

Hmm, I haven’t ever washed DNA with ethanol before, that’s strange. Well, columns I have, but not a pellet. In general, though, you need to get rid of all of it. Ethanol will disrupt most reactions you’d want to do with DNA. For getting rid of it, blowing air is probably the best method, since it will evaporate readily, but you’ll have to be careful to make sure it really it fully gone.

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