I am trying to do a gene expression assay and I have to wash the cells in starvation media. I am trying to use a centrifuge instead of a filter to save a lot of money. What is the best way to spin cells down so I get a good pellet, that wont come loose, without killing the bacteria. Please respond in RCF or Gs.
I am using starvation media to investigate PhoP regulated genes as previously described (Bougdour, 2008). However, in this paper they used a filter to collect the cells from the broth and to wash the cells. In my lab we only have disposable vacuum filters and they are not cheap. So I am trying to find the fastest way to collect the cells using a centrifuge without killing cells. I am using gram negative bacteria. I have heard and read that spinning cells too quickly and too long will kill some of the cells in the tube. I am using a 50 ml falcon tube to spin 30 ml of cells at 0.3 OD. My big centrifuge has a max speed of 20,133 rcf. It would be nice if I could get a tight enough pellet here that I could decant the sup without losing too many cells. I have used 20,133 rcf for three minutes to pellet cells.
I then suspend the pellet in 1 ml of starvation media and transfer to a 1.5 ml microcentrifuge tube and spin again in a smaller centrifuge at 20,133 rcf for three minutes. I then wash them again in the same way. Once I resuspend the pellet I put them in the incubator until 50 minutes after the first wash.
I was able to do qPCR on these samples and got results that are consistent with PhoP regulation but I have to do this experiment several times and with different cations.
I want to make sure that I am not potentially skewing my results by spinning the cells too fast. My expression was normalized with 16s.
Thank you,
Austin Wright
Dear Austin,
I have spinned several bacteria with 5100-5530 RCF without killing them. Important is the tube it should be konic not round.
If you have problems to convert RCF in RPM use this help.
http://www.hettweb.com/mobile-app
First thing during centrifugation bacterial cells will not die.
Please provide your protocol because under normal centrifugation conditions irrespective of the shape of bottom of tube the vegetative cells can be recovered from the pellet of the tube.
Are you performing some kind of washing with simple water? If yes then it would be the culprit. The bacterial cells would have died because of the hypotonic solution.
Please elaborate your protocol...
I think you must be spinning it at around 4*C if not it is having any cold-shock response.
I am using starvation media to investigate PhoP regulated genes as previously described (Bougdour, 2008). However, in this paper they used a filter to collect the cells from the broth and to wash the cells. In my lab we only have disposable vacuum filters and they are not cheap. So I am trying to find the fastest way to collect the cells using a centrifuge without killing cells. I am using gram negative bacteria. I have heard and read that spinning cells too quickly and too long will kill some of the cells in the tube. I am using a 50 ml falcon tube to spin 30 ml of cells at 0.3 OD. My big centrifuge has a max speed of 20,133 rcf. It would be nice if I could get a tight enough pellet here that I could decant the sup without losing too many cells. I have used 20,133 rcf for three minutes to pellet cells.
I then suspend the pellet in 1 ml of starvation media and transfer to a 1.5 ml microcentrifuge tube and spin again in a smaller centrifuge at 20,133 rcf for three minutes. I then wash them again in the same way. Once I resuspend the pellet I put them in the incubator until 50 minutes after the first wash.
I was able to do qPCR on these samples and got results that are consistent with PhoP regulation but I have to do this experiment several times and with different cations.
I want to make sure that I am not potentially skewing my results by spinning the cells too fast. My expression was normalized with 16s.
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