Low yield in maxiprep - (Oct/11/2006 )

Hi

I am getting very low yield using a quiagen maxi prep whereas it gave very satisfying yield for different plasmids. Can anybody help me giving some really useful tips about increasing yield.

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Might this low yield plasmid be a low copy number plasmid (but still within the range of column binding)? If so, simply increase the number of maxiprep cultures for that plasmid. Double the number of culture flasks that you are using.

If the plasmid is a high copy plasmid or one whose size lies outside the column binding range, hehe.. i am afraid that you have no choise but to do the old tried and true method of alkaline lysis.

You would find this method is cheaper and has no upper limits on the quantity of plasmid extracted. It will happily extract 1ml as it would 12 litres. So low yields... just grow more cells. (Yes... this method is far slower.)

Finally you can use better media to grow more cells see topic
http://www.protocol-online.org/forums/inde...?showtopic=1663

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As Perneseblue said, it could be a problem with the copy number of the plasmid. In that case, he is right in saying that you need to grow more cells. You can do an alkaline lysis of a larger culture volume and then use the column from the Qiagen kit to give you a more cleaner plasmid yield. I also have found that a major problem with the kit is DNA precipitation. The pellet is easily lost so you need to be very very careful. I tend to precipitate and then collect the DNA in 2 ml eppendorf tubes. I then use 100-200 ul of water to dissolve the DNA by passing the water from tube to tube.

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if u have a low yield plasmid, grow a day culture (10ml) and use it to inoculate the maxi culture O/N (500ml).

Use a single column from the kit and u should get a good yield.

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thanks for all these tips. I would like to combine them and perform today. lets see what happens. gonna let you know people.

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