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Base pairs apart between 2 sites for sucessful double digestion? - (Jan/18/2007 )

How many base pairs apart between 2 restriction sites are sufficient for sucessful double digestion? If there are 12 bp apart for NheI and XbalI, is it OK? Thanks!

-plant-

12 bps is more than sufficient.

-scolix-

12 bp is sufficent in your exp.
For such purpose, you may have a look at neb webpage "cleavage close to DNA ends". Some enzymes need more than 6 bases some are ok with just2. So that may be helpful for sequential digestion.

-fred_33-