cloning genes with incompatible restriction sites - Protein expression (Jun/01/2005 )

Hi,
I am trying to clone and express a protein into the pET-30 but I have an issue here

1. The gene is cloned in a vector and flanked by Not1 at the 5' upstream of the gene ORF & Sal at 3' downstream, and it will be cut from those sites off the vector (pCMV- vector).

2. Once the gene is cut, I want to digest it by those restriction sites and clone immediately into the pET-30 which will be also digested
from the same sites.

The problem is that orientation of those sites (Not I and Sal I) in the pET-30 vector (expression vector) is REVERSED. The Sal I is upstream and the Not I is downstream. This means if I clone it this way the ORF of the gene will be down steam at the Not1 sites (reversed) and will not expressed.

I do not want to do PCR because I tried to express the gene 3X and did not work even with proofreading Taq.

Any help will be appreciated
MoleMicrobiol

hi
i would suggest you to buy two oligos that kills the restrictions sites and contain the 2 restriction sites in the good sense... respecting evenually the ORF of your gene.

Fred_ 33,
Thank you very much for the help.

MolecMicrobiol