I want to design a polylinker sequence to be inserted into a vector.
Should I provide overhang prior to each restriction enzyme, as I have done in this?
Or overhangs at 5' and 3' is enough?
5' -AAAGAATTCTTTCTCGAGATACCGCGGCCTTTAATTAAAAAGGCGCGCCCCGGTCGACTTT- 3'
(Nucleotides in bold - Restriction enymes; Regular - Overhangs randomly inserted)
Hi there,
There is no absolute need to introduce spacer sequence between restriction sites unless you fear the effect of immediate proximity of sites in a double digest. If you look at MCS of pUC19 for instance the sites are side by side and even overlapping sometimes.
https://www.neb.com/products/n3041-puc19-vector
if confirmation is useful I completely agree with Dominique.The other RE sites provide additional bases and the need for extra bases only really applies to cut sites close to the end of linear dna fragments/amplimers
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