In article <Pine.SOL.3.90.960213151216.24019A-100000 at uxmal>,
edgar at cifn.unam.mx (Edgar Valencia) wrote:
> 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
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.
"The two most common things in the universe are hydrogen and stupidity" Harlan Ellison