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Using more transformed bacteria for midiprep


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#1 science noob

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Posted 11 September 2014 - 04:18 AM

How much extra can we go in terms of amount of bacteria used to purify a plasmid of interest?

 

Most protocols recommend 25-30ml liquid cultures but has anyone tried pelleting more bacteria to get a higher plasmid yield especially for larger constructs.

 

 



#2 Rsm

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Posted 11 September 2014 - 07:21 AM

Which columns are you using? The Qiagen Giga kits recommend 5 liters of culture volume. For a Midi I usually use 50ml, Maxi 100-200ml... Depends on your columns, and how much DNA they can bind.


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#3 seanspotatobusiness

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Posted 11 September 2014 - 12:51 PM

OMG, you noob! GTFO!

 

Measuring the amount of bacteria processed by how much culture medium you're using is a very rough way to work. I think it's how my colleagues do it but I have a slightly less rough approach using the Sigma GenElute Maxi Kit. I measure the OD600 (for E. coli, usually DH5alpha) and then divide 450 (given in manual) by the OD600 to determine how much culture to process. You may have to contact the manufacturer of your kit to find out the optimum mass of bacteria. This can change depending on your strain and plasmid but my (wild?) guess is that you don't need to worry about that.

 

If you try to process to large a mass of bacteria without adjusting the volumes of lysis and associated buffers, your yield may go down because so many bacteria will not have been properly lysed.

 

I don't know whether there are any drawbacks to overloading a column apart from wasting the excess plasmid.


Edited by seanspotatobusiness, 11 September 2014 - 12:51 PM.





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