sara_t at my-deja.com wrote:
> 1- I checked the enzimes I used, but before doing the REAL digestion of
> the plasmid; in fact, I inserted the restriction sites into the promoter
> by using primers containing the sites sequences, by PCR. So, the enzimes
> would have cut a few number of bp, undetectable by agarose gel. INdeed, I
> had to TRUST the digestion was good. I made it overnight to be sure of
> this.
When you designed your primers with the restriction sites on them did
you put a few extra bases after the site, so it wasn't right at the end
of the primer?? Many restriction enzymes will not work properly if
there is no flanking sequence after the site (I don't have the data for
Sac / Hind to hand). Whenever we design such primers we always put 6
extra bases (CATCAT - but anything sensible should work), after the site
on the primer.
> 3- The molar ratio I used was about 2/1 for the promoter (maybe this is
> wrong).
A ratio of 2:1 shouldn't stop your ligation from working, the worst you
would get is reduced efficiency.
> 4- I purified the insert by gel purification and elution with "ultrafree
> DA- Amicon". But this gave me bad results, so the last time I didn't
> purify the fragment at all, I achieved it by PCR and it was very clean.
Are you doing some clean up after the PCR though? You shouldn't just
add restriction buffer to a PCR mix and hope that it will work - there
will be salts in the mix from the PCR reaction. You should at least
ethanol (isopropanol - whatever!) precipitate and resuspend before
restricting.
Hopefully one of these will point you in the right direction!
TTFN
Simon.