Sv: Inserting PCR fragment with SfiI?

Jens Tornoe jens at tornoe.net
Thu Jun 8 14:04:17 EST 2000


Hi Jesper

In most cases, 4-5 bases is more than enough to protect your sites. I never
worry too much about the NEB chart, but take it more as a rough guide. For
enzymes not found in the chart, I routinely add 5 bases. It usually works,
so your 6-base protection is probably fine, I would say.

Good luck!

Jens Tornøe
NsGene
Ballerup, Denmark


Jesper <jsp at imsb.au.dk> skrev i en
nyhedsmeddelelse:393FAD01.7D5DDAD9 at imsb.au.dk...
> 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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