Help PCR product ligation problem

[upyers]

In article <Pine.SOL.3.90.960213151216.24019A-100000 at uxmal>,
edgar at cifn.unam.mx (Edgar Valencia) wrote:

> Hi 
> I'm having problems cloning a PCR product
> The reaction seems OK. The idea was to obtain the product using oligos 
> with the EcoRI site at 2 pb from the 5', after digesting with EcoRI to clone 
> the fragment into the vector im interested in.
> My procedure consist in 
> Do PCR reaction
> Cleaning the product using Sentrisep columns
> Extracting with phenol-chloroform-isoamylic alcohol as well as with 
> chloroform like in alkaline lysis
> precipitation with EtOH 100% and washing with EtOH 70%
> Dry the pellet
> digesting overnigth with EcoRI
> Extracting again with phenol and chloroform
> and reprecipitate
> do the ligation reaction
> Transform the cells
> I cant obtain ligation of the PCR product against itself. 
> As far as I know, EcoRI doesnt have problems cutting at the edge, and my
> oligos have 2 pb at 5'
> Any suggestion?
> Thanks in advance
> 
> Edgar

Edgar;

Unless your target is inherently unstable such as repetitive DNA, or it is
a retroviral target (notoriously unstable) I suggest you digest your PCR
product directly after cleaning it on your column.

> might I suggest an affinity column like the Qiagen PCR product
purification system, although barrier type thingies like Centrasep column
often work fine<

Don't extract/precipitate it. You can then run the digested product on a
gel and excise the band of the correct size (or do a in-gel ligation) for
your ligation rxn. 

I suspect that the various manipulations with organics that you are doing
with the DNA are at fault here.

Good luck.

-- 
"The two most common things in the universe are hydrogen and stupidity" Harlan Ellison