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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!


12 bps is more than sufficient.


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.