I am expressing a construct in E. coli under different conditions to check soluble expression. I have a large array of conditions, which cannot be done in flasks. Thus, I will be growing cultures in deep well 96 well plates. After cell lysis, I need spin down the plate to separate sup and pellet. This generally requires spinning at speeds >10k x g. However, the swing bucket rotor that has plate adapters can spin at 5k x g at the highest. How can I separate sup and pellet at this speed? Is it possible to achieve separation by spinning for long duration (>1 h)?
I think it works, I pellet the plasmid by 5k for 1 hr, it works as same as 13k for 15min.
to some extent, yes, this is a function of g x time and you can get a pellet by spining longer
https://www.coleparmer.com/tech-article/basics-of-centrifugation
if you are doing cesium prep of plasmid like in 1980th in very dense medium you will never get a pellet. but in this case spinning longer is the answer. you just have to be careful as pellets are a bit loose since they were generated by smaller g-force
My experience is that with 1.5 ml Eppendorf tubes, spinning for 2 min at 15 000 g is enough to pellet E. coli cells, so with 5 000 g, 10 min should be plenty, particularly since the length from meniscus to bottom is much shorter. However, as pointed out by Igor, the pellet is likely to be a bit looser.
Formally, you can use the pelleting efficiency k of the rotors to calculate the time required from k1/t1 = k2/t2. The k-factors should be in the rotor spec-sheets.
Vihang Ghalsasi
As Pierre Béguin mentioned, you should use eppendorfs. I our studies' I had used eppendorfs and those were over 600 samples to do the GC analysis.
Dear Vihang
It depends how clean have to be your surnatant.
In my experience plate centrifugation for 1h at 3000g which work in eppendorf and thermo centrifuges with plate adaptors is able to remove the most of the e.coli cell dedries and you can use the surnatant for IMAC or other purification with gravity flow coloumns.
Otherwise if you need to remove all the cells, you can perform a 96 well plated based 0,45 or 0,22uM filtration on the collected surnatant but the plates are quite expensive.
Good luck
Manuele
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