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"Easiest" sticky end combination - (May/21/2013 )

All else being equal, what two restriction enzymes when used together yield the insert that is easiest to insert (single copy) in the correct orientation?


I like the Biobrick choice of EcoRI and PstI. Both are cheap, reliable, can be heat killed, work in the same buffer, are insensitive to E. coli methylation, and are available in high fidelity versions from NEB. They produce different overhangs: EcoRI a 5' overhang, and PstI a 3' overhang, so mis-ligation is unlikely. They also both cut near the end of dsDNA fragments with only a few bases outside the RE site.


I 'll go with the combination of enzymes that has higher cleavage efficiency (towards the end of oligos) in short time as per NEB like Bam HI, ECORI, Kpn and Xba (ofcourse there are some more), besides Hind III, Not1 are also easier....


My favorites are EcoRI, BamHI, and Xba1