Plasmid isolation tips

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Contributed by Dr. Alexei Gratchev

Ampicillin concentration in bacterial culture

Working concentration of ampicillin usually indicated in laboratory manuals is 50 or 100 µg/ml. However the cultures with standart high copy number plasmids, such as pUC19 or pBluescript can grow at much higher ampicillin concentrations resulting in higher plasmid yield. The highest concentration I have used was 1 mg/ml! I would not recommend to use such an extreme conditions for your culture, but increase of your standart working ampicillin concentration to 200-400 µg/ml will already increase your plasmid yield 2-5 fold. For example from 1.5 ml overnight culuture supplemented with 300 µg/ml ampicillin I was able to isolate up to 50 µg plasmid using standart 3-solutions protocol.

Increasing the plasmid yeld from a single column

Plasmid isolation using silica matrix columns is a very popular method. Such columns are avaliable from different companies (for example Qiagen). The capacity of the column is usually limited. However there is a possibility to double or even triple you yield by using the column 2 or 3 times for the same sample. As it is stated in the manual for Qiagen columns, the silica matrix is stable for several hours. Therefore the only thing you have to do is to prepare double amount of culture, split your lysate into two fractions and isolate plasmid from the first fraction using standart protocol. After the elution the colum can be equilibrated again and will be read for the second fraction of the lysate. Depending of the speed the solution goes througth you can purify 2-3 fractions on one colum.

Increasing the speed of plasmid preparation with Qiagen or similar silica matrix columns

If you isolate your plasmids with Qiagen or similar silica matrix columns, you know the problem that some of the columns run faster, some slower and some very slow. To solve this problem you can attach a 2-3 cm piece of silicon (or plastic) tubing on the tip of the column as shown on the figure. This will increase the speed of gravity flow of the column up to 3 fold, during binding and washig steps! It's better to remove it while eluting the plasmid, to keep the elution efficient.

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