Inserting PCR fragment with SfiI?

Jesper jsp at imsb.au.dk
Thu Jun 8 09:25:46 EST 2000


I would like to clone a degenerate PCR fragment into a vector using SfiI
and KpnI. The primers that I use for the PCR contains SfiI and KpnI
sites, and I had no problem designing the KpnI-primer since only a 2-3
bases flanking the site is necesary for >90% restriction (eg :
cggGGTACCccg - source: NEB catalog 96/97). But I have had problems
finding similar information about SfiI, so I have constructed the
SfiI-primer to have 6 g/c basepairs flanking the SfiI site
(5'ggcggcGGCCcagccGGCC---). Would this be okay, or should i have more
bases flanking the site ?

Sincerely Jesper S. Pedersen


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