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Question making competent bacterial cells - (Aug/28/2007 )

Hi,
I wonder if I have to use overnight grown culture before I grow them in a large volume.
Can I just use frozen bacteria in a large LB culture to get OD600 of 0.4?
Thank you for your answer in advance!

-bara-

Yes you could do that, just make sure that the bacteria are clonal (i.e. you have grown the culture from a single clone) and that you use a sufficient amount of bacteria, otherwise it could take a long time to grow. A good approach is to make a cell bank - grow the cells to mid log phase (OD=0.5 or so) then freeze 1-2ml aliquots in glycerol at -80°C. When you want to grow up a culture for competent cells, just innoculate with one of your aliquots (use a 1% innoculum, e.g. 1ml in a 100 ml culture) and you should get an OD of 0.4 or so in a few hours, depending on the growth conditions.

-bitesizebio guy-

Great! Thanks a lot!


QUOTE (bitesizebio guy @ Aug 28 2007, 09:42 AM)
Yes you could do that, just make sure that the bacteria are clonal (i.e. you have grown the culture from a single clone) and that you use a sufficient amount of bacteria, otherwise it could take a long time to grow. A good approach is to make a cell bank - grow the cells to mid log phase (OD=0.5 or so) then freeze 1-2ml aliquots in glycerol at -80°C. When you want to grow up a culture for competent cells, just innoculate with one of your aliquots (use a 1% innoculum, e.g. 1ml in a 100 ml culture) and you should get an OD of 0.4 or so in a few hours, depending on the growth conditions.

-bara-