Adding extra bases varies according to the kind of restriction enzymes you use. Many scientists advise to add 3-6 nucleotide upstream enzyme recognition sequence. If I did not add extra bases, Will I face poor digestion for my cloning?
Hi Abdou
Yes you will. Even if you did not add the proper number of nucleotides (based on the enzyme) you will have poor cut. I faced this before.
Dear Abdou
i would say yes it will affect, since many restriction enzymes do not cut DNA efficiently at the end of a linear piece, so add 3-6 bases should improve cutting efficiency. you can chick : https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments#.UEecadBWrDY
also,
https://www.addgene.org/plasmid-protocols/pcr-cloning/
good luck
Ahmed
Yes, it will make the restriction more difficult. We normally add the restriction enzyme in portions in these instances, the first addition in the beginning and then add after 30 and 60 min also.
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