Help PCR product ligation problem

Edgar Valencia edgar at cifn.unam.mx
Tue Feb 13 16:32:16 EST 1996


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



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