I have an DNA insert that I intend to order as a complete double stranded construct and clone into a vector using restriction enzymes, however I am unsure exactly how to design the restriction sites that flank the insert. Currently I have the restriction sites right on the ends of the insert, that is, there are 6 bases stuck on each end, and the site is undigested/ not 'sticky'. Are restriction enzymes (BamHI and XhoI specifically in this case) able to cut this, or do they need DNA on both sides a restriction site to recognise/ cut it?
Thanks
Most type II restriction enzymes work best if there is some DNA on either side of the recognition site. They will work at low efficiency at the very ends of the DNA, but often not sufficiently for a cloning reaction. Usually five or six extra bases beyond the restriction site is sufficient to get good digestion. A low complexity sequence (e.g. ATATA) is fine to add to the end of a sequence to facilitate restriction digestion.
Hi,
see the overview here
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
Best Sven
As Nicholas J Harmer mentioned you need to add more bases outside the sides as different enzymes require a different number of bases outside the restriction site to digest properly. This is been characterized for most enzymes (https://international.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments). I personally always add 5 additional bases on each side just so I don't have to check the specificity of each enzyme all the time.
Good luck!
Hanna
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