FAQ: What is the minimum distance needed between two SfiI sites? Can they follow each other directly?

This question can be answered at a few levels. If you merely want to achieve an efficiency of digestion as good as in a vector having a single SfiI site (which is an inefficient digestion) then you can use a separation of approximately 10bp. For optimal efficiency of cleavage of the two SfiI sites, you would want to include a ""stuffer fragment"" of at least 170bp. [I would suggest 200bp.] The kinetics of SfiI appear to be unique among commercially available restriction enzymes. For this reason, SfiI reactions can be difficult to optimize. A vector with a single SfiI site is probably the most difficult type of SfiI site if the goal is to achieve absolute completion. Both the ratio of enzyme: DNA (units:micrograms) and the DNA concentration are important. A vector which has two SfiI sites is cut much more rapidly, due to a cooperative reaction involving both sites, BUT ONLY if the the two sites are separated by at least 170bp (enough to allow looping of the

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