I need to digest my plasmid with 2 or 3 different restriction enzymes. Can I perform a simultaneous double or even triple digestion?

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Answer

If you are able to find a buffer in which all 3 enzymes have sufficient activity (usually not lower than 50%), you can set up a single digestion with all 3 enzymes. It is important that the total volume of enzymes you add to your reaction is not more than 1/10th of the total reaction volume. The reason for this is that some enzymes have star activity if the concentration of glycerol exceeds 5%. If you are not able to find a buffer in which all your enzymes have sufficient activity, you will have to perform sequential digestions of the plasmid with the individual enzymes.

Answer Id: E9848

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