Ecor1/Pst1 digest - (Jan/22/2008 )

Hi Everyone
I am cloning with Ecor1/Pst1 and was wondering what is the best way to set up this digest----is there a way I can do a double digest or do I have to do sequential----and how do I incorporate Ecor1 unique buffer to minimize star activity?
thanks
Lisa

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I would use 2x as much PstI as EcoRI and double digest in EcoRI buffer + BSA for 1-2hrs. Make sure your reaction volume does not have more than 5% glycerol.. ie, use 0.5uL EcoRI, 1uL PstI, in a minimum of 30uL.

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We do this double digestion routinely in NEB buffer 2 + BSA. Both enzymes cut very well, and quickly. As Judes said, avoid high amounts of glycerol by limiting total enzyme mixed into the reaction. If you can, do the digestion relatively dilute, with 1 ug in 50 ul or so, with 1 ul of each enzyme. NEB has stopped shipping a custom EcoRI buffer.

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thanks Judes
Lisa

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Thanks Phage 434
Lisa

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