Centrifuge - Want to know important principles (Apr/14/2009 )
I dunno where I should post this so i think that this is the best place..
I use centrifuge very often and I would say that many other researchers also used it in their experiments. However, I only use it based on the protocols that have been written. I would like to understand the principles of centrifugation so that I can use it without following the protocols blindly and determine my own parameters when carrying out my own research.
My questions are:
1) I understand that the in centrifugation, the heavier product will be sedimented as the pellet while the lighter product will stay as supernatant. There will be a certain minimum threshold to the size/density of the particle to become the pellet. Insufficient time or centrifugal force will result in more components to stay in the supernatant while excessive time or centrifugal force will make you lose your desired product to the pellet. How do you determine the time and centrifugal force (g) to be used to obtain your desired product in the supernatant/pellet?
For example in a paper, one of the step is
"Samples, 10 ml, were centrifuged for 5 min at
5000 g. The biomass pellets were washed twice and
then dried in an oven at 110 ◦C to constantweight. The
culture supernatant was analysed for glucose using a
commercial kit (Sigma)."
How did you come to the parameter 5 mins as the time and centrifugal force as 5000g?
2) The second question, is somewhat similar to the first question. In an experimental design requiring centrifugation as one of the methods, what is the best way to determine the parameters ( time, centrifugal force) to get the an uncharacterized product in the supernatant so that the minimum size in the supernatant is my product where bigger molecule will be sedimented as pellet?
Hopefully someone understand the principles and share wif us. thanks
you can get information on centrifuge theory and practice (including equations) in the literature that ships with the centrifuge.
you can also get literature from beckman-coulter at this webpage:
centrifuge literature
Thanks dude,, A lot of information there... I think it should help...
My quick summary (not applicable to ultracentrifuges!):
It doesn't much matter except for breaking things. 2000-3000g for 96 well plates. 6000g or so for thin wall pcr tubes (you will be unhappy when your precious sample goes into the rotor). 8000g for most conical 15 and 50 ml tubes. 15000g for eppendorfs, including column minipreps. Final elution of miniprep columns into capped tubes can be done at 6000g if you want to keep the loose caps on the tubes.
phage434 on Apr 16 2009, 04:20 AM said:
It doesn't much matter except for breaking things. 2000-3000g for 96 well plates. 6000g or so for thin wall pcr tubes (you will be unhappy when your precious sample goes into the rotor). 8000g for most conical 15 and 50 ml tubes. 15000g for eppendorfs, including column minipreps. Final elution of miniprep columns into capped tubes can be done at 6000g if you want to keep the loose caps on the tubes.
How about 30 000g?? It was used in isolation of unnown growth factor from bacteria.. the g u mentioned are all small compared to it
Most benchtop centrifuges can't reach 30,000g. Few protocols require it that wouldn't work better at ultracentrifuge speeds.
phage434 on Apr 16 2009, 08:10 PM said:
Im not sure about this cuz I am an amateur researcher but in articles concerning isolation of compounds from bacteria usually they use around 30 000g. Maybe they use different kind of centrifuge. Anyone done this kind of research before? Can you confirm about this or is it just the paper that i read is an exception??
But i wonder how they come up with the time and rcf to be used..
As you very well known the principle of centifugation,the time and centifugal force depends upon the matter and the quantity of that matter.If we are centrifuing the very fragile protein or something then we have to keep the time and force as low as possible and vice-versa.
You can ask your question and clarify all your doubts by discussing with the teachers of WiZiQ they will help you in everything.You can go to biology tutor for more help.
Regards
Rupam
c0ok1e on Fri Apr 17 05:46:50 2009 said:
Most benchtop centrifuges can't reach 30,000g. Few protocols require it that wouldn't work better at ultracentrifuge speeds.
Im not sure about this cuz I am an amateur researcher but in articles concerning isolation of compounds from bacteria usually they use around 30 000g. Maybe they use different kind of centrifuge. Anyone done this kind of research before? Can you confirm about this or is it just the paper that i read is an exception??
But i wonder how they come up with the time and rcf to be used..