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Sequential digestion - Sequential digestion using two RE with different incubation temperatures and dif (Oct/03/2005 )

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How to use 2 enzymes with different buffers?

I have HpaI (50% in buffer2; 100% in buffer4) and SpeI (100% in buffer2) to digest genomic DNA. Lets say if I would put HpaI in mixture solutuion before 3 hrs upon I will add SpeI and store o/n would it be good trick or I could mix both enzymes at the same time?


if the difference is that large, I usually call up NEB's tech support and whine till they give me a protocol for such a digestion

I have had good luck this way


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