In article <200005022020578.SM00469@[216.220.170.54]>,
jaymone at paonline.com ("Jay Mone") wrote:
> A couple of questions about controls you are running during your procedure:
>> (1) Have you checked to see that your enzyme is indeed cutting the plasmid?
>> (2) Are you dephosphorylating the plasmid to help prevent self ligation?
>> (3) What is the molar ratio of insert to plasmid? It should be about 4:1
>> (4) How do you purify your insert? Gel purification or something else?
>> These are simple things, but from the info you gave, I'm starting from
> scratch.
>> Jay Mone'
> ---
> I can quickly answer:
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.
2- I haven't dephosphorilated the vector for the ends are uncompatible
(sac and hind cut).
3- The molar ratio I used was about 2/1 for the promoter (maybe this is
wrong).
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.
Surely I've made some mistake during one of these steps, because of I' m
young at this work...But I repeated all the sequence of steps three time,
trying to be more careful.
Anyway thank you very much, I'll make you know my eventual success!!!
Bye to everyone of the group Sara
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