I am designing a primer for a gene having BsaI restriction site. Do I need to include 3-4 base ahead of recognition site for enzyme to bind.
Hi Akshay, it looks like BsaI needs at least one base ahead of the restriction site (if the recognition site is near the end of the DNA strand). But it also works just as well with 2-5bp ahead. Here's the link from NEB I got the information from:
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
Hope this helps!
Hi there,
As NEB hasn't actually tested the enzyme activity in case of no added base pair upstream the cutting site, one can't conclude about the absolute necessity of at least 1bp for BsaI...
Yes, I would do that. It cannot hurt to have a few extra nucleotides before your restriction site. Generally, enzymes do not work well if the recognition site s at the extreme end of a fragment.
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