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ideal speed (rpm) for harvesting pellet for protein expression

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zepedrof
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Topic Started by zepedrof
on 8/25/2009 13:26 PM   
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What is the ideal speed for harvesting 400 mL of bacterial culture from induced pellet ( 1mM iptg) ?

Some protocols say 4000 rpm at 20 minutes or

others at 14000 rpm at 20 minutes ?

I obtained my pellet centriguginf at 14.000 for 12 mins... would that be ok ?

is there any difference from a 14.000 rpm pellet from a 4.000 rpm pellet ?

cheers,

JP


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Pippuri
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Posted By Pippuri
on 8/25/2009 13:46 PM   
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 Hi Zepedrof,

I think you will get many answers based of individual's preference.  (Having said that, I personally prefer 6500xg, 15 min, 4 deg....)

I don't think using either of the conditions is going to harm your experiment outcome per se. The only thing that I could think of is how tight the pellet will be packed under different centrifugation force, and it may "remotely" relate to how quickly you can resuspend the pellet for your next step...

:-)



Suola
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Posted By Suola
on 8/25/2009 13:51 PM   
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zepedrof,

In order to pellet cells, people usually use around 5000 g (centrifugal acceleration 5000 times the gravity on earth) for 10 minutes.

You achieve this g value at different rpm values depending on the size (diameter) of the rotor. Also, to an approximation, you can shorten the time to half if you double the g value and get the same result.

Thus, the rpm and time in different protocols can be different even when using the same g value if the protocol uses different rotor.

The lesson is that convert the rpm value to g value when comparing protocols. Your centrifuge manual usually tells the relation between the rpm and g values for different rotors.

The idea of harvesting cells is exactly what it sounds, if after centrifugation, you see a nice tight pellet and clear supernatant, your protocol worked. Thus, there is no ideal speed, as long as it pellets the cells visibly.

Cheers,

Certainty of death, small chance of success... What are we waiting for? - Gimli


Last edited Oct 27, 2009, 6:23 AM by Suola


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Posted By Add colour 2 ur life
on 10/27/2009 4:01 AM   
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Hi,
I am there with pippuri, as he said you might end up with hard pellet if you go for more speed.For pelleting the cells i dont think you need not more than 5000X g.And i dont think you will not have any difference with induced bacterial cells over  un induced cells, you can use the same parameters.


All the best.

Balu...




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Posted By yashwant_chavan
on 6/26/2013 19:55 PM   
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4000 rpm for 10 minutes is enough to pellet bacterial population in 1mL sample collected in 1.5mL eppendorf tube. Depending upon the type of sample you are using the final outcome will vary if you use higher centrifugation speed. If the sample is environmental water or any biological fluid containing particles and biological content other than bacteria also then you would end up in pelleting other particles having pelleting speed greater than 4000 RPM. Hence to precisely pellet bacterial population 4000 rpm for 10 minutes is enough.



ajsr1987
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Posted By ajsr1987
on 10/30/2014 14:21 PM   
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Hi there,
I know its a bit late to reply to the thread, but I found this article, which suggests that using too high centrifuge speed may actually cause damage to the cell surface and its interior structures, resulting in lower viability and impaired function. Please check this out before deciding on the appropriate centrifugation speed.
http://aem.asm.org/content/early/2011/10/24/AEM.06780-11.full.pdf



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