Adaptor for salI-EcoRI - Adaptor compatible for SalI and EcoRI (Apr/10/2005 )
Can some1 suggest where can I get BASIC INFO about designing an Adaptor for ligating my 3.5 kb insert with EcoRI and SalI ends into Vector cut with EcoRI.....Quick guidance will be really treasured.Thanx
NEB web site will helps great on this question.
But you need an adaptator with both sites. Let say enzyme A and B
first, regarding one enzyme at time, it can't cut if restriction site is directly at end of fragment. Hence you need to add one ore two specific base for increasing restriction efficiency (Cleavage Close to the End of DNA Fragments neb's webpage) (but the previous version of site used to tell wich bases should be added ; it's in their green 2004 catalog)
you further need to add 2-3 "dummy" bpairs in order to separate the two restriction sites.
But for your problem, i would suggest to order two oligos. The priinciple is to cut your vector by EcoRI. The ligation of insert which would contain EcoRI and SalI sites should "killl" the EcoRI site.
Hence if i suppose EcoRI digest would make these ends:
your fragment should be
Hence in the ligation the original EcoRI site will be killed.
the two restriction sites should be separate by at least CGGAGACGC (based on informations given by neb).
Hence your oligo should be
1:AATTT CG GAATTC GAG ACGC GTCGAC A (eco site folowed by sal I site)
2 : T GTCGAC GCGT CTC GAATTC CGA
Just anneal these two oligos and insert them in te cutted vector (annealing tips here).
The information u provided was really helpful. Finally I have designed an Adaptor for this ligaiton reaction. Thanx a lot. (NEB data was really handy)