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Distance between RE sites for double digestion - (Sep/15/2011 )


I want to double digest a pCI-neo plasmid with SalI and NotI, but there are only 4 nt between the RE sites. Will they double digest successfully? I remember somebody told me that they won't because the RE which binds to the first site won't give enough room to the next RE to bind and digest. true?


hmm, I would say you could try. Usually it is 3 to 6 nt flanking the restriction sites, and for some enzymes it goes up to 8 nt. In my previous lab we had an old NEB catalog with a list of number of nt necessary for the enzyme to cut. (and plenty of good informations too). Now I have some difficulties to find the information.

-little mouse-

yes, the problem is the only two RE sites not cutting the gene of interest are SalI and NotI. All other REs in the MCS site cut the gene of interest. unless I do single digestion, first NotI and then SalI. If I cut with SalI first there will be no overhang nt left for NotI to bind.


very naive question, did you check if you have sites in your gene of interest with compatible ends with some other sites from the MCS?

-little mouse-

yes, i did.


New England Biolabs has some great literature for this and here is the link:

I looked at pCI-Neo, and you shouldn't have any problems with this double digestion. Both SalI and NotI will cut just fine with 4 bases to the end of the fragment. Remeber, both enzymes don't have to cut simultaneously, one will cut first and the other will cut the linearized plasmid.

Best of Luck.