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Sonication issues!


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#1 S.A.P

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Posted 01 March 2010 - 04:46 AM

Following sonciation of my cells, I centrifuged as normal to harvest the insoluble protein fraction. Only to find I couldn't get a significant pellet no matter how much I spun the samples down!

Any ideas? Perhaps the sonication time was excessive?

#2 lab rat

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Posted 01 March 2010 - 09:22 AM

How long did you sonicate, and did you have the sample on ice?
42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.

#3 scolix

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Posted 01 March 2010 - 10:31 AM

What buffer did you sonicate the sample? If the insoluble protein is extracted by the buffer, I guess you might see any pellet.

#4 S.A.P

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Posted 02 March 2010 - 02:49 AM

I always sonicate at a 1:3 ratio (5 mins actual sonication time, 15mins off) per pellet from 200ml of culture. My samples are always kept on ice during the procedure too. The amplification was 40%.
I was trying two different buffers, one was something I have always used (tris, DTT, EDTA and NaCl at pH 8), the other was bought from Qiagen. But I didn't get a pellet for either once I had spun them down!

I've never had this problem before but I guess I will soon find out if this really matters soon enough!




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