4 replies to this topic

[sxh999]

Hi everyone,

I wanna store my bacterial cells before extracting DNA, so I usually centrifuge 1.5 mL sample at 10,000 rcf for 10 minutes to concentrate the cells.
Unfortunately, I was told that 10,000 rcf was too high and that would damage the cells.

Could anybody who has this kind of experience please tell me the truth or some information? I could not find anything information about that.

Thank you everybody.

[phage434]

I don't think 10,000 x g will harm bacterial cells. I'd be more concerned about osmotic stress from changes in the culture medium and freeze/thaw in the absence of cryoprotectants.

[sxh999]

phage434, on 03 May 2011 - 06:32 PM, said:

I don't think 10,000 x g will harm bacterial cells. I'd be more concerned about osmotic stress from changes in the culture medium and freeze/thaw in the absence of cryoprotectants.

Thank you so much.

I usually centrifuge the samples at 4 degrees and 10,000 rcf for 10 minutes, then discard the supernatant. There should be pellets on the bottom of the centrifuge tubes. Then store the tubes with pellets in -80 degrees for 2-3 days prior to extract DNA.

When start extracting DNA, take the tubes out and put them in ice box, then follow the DNA extraction protocol.

Do you mean that the 10,000 rcf centrifuge force won't tear the bacterial cells but the significant temperature changes will impact the cells?

Thank you again.

[phage434]

You're planning on lysing them after thawing them in any case. The thaw will help.

[sxh999]

phage434, on 04 May 2011 - 12:46 PM, said:

You're planning on lysing them after thawing them in any case. The thaw will help.

Thank you for your help.

I think so. When it thaws, the cells lyse, then it would be beneficial for DNA extraction later. However, when the cell density is very low, after 2-3days storage in -80 degrees, it is really hard to extract DNA from that sample, I don't know why.

Thanks again.