Plasmid frameshift problem after clonning - (Mar/10/2010 )
I've got a v. basic clonning question here. I've inserted a full-length gene into a pET15b vector. When I got my sequencing results, I found the reading frame of the vector is changed. The plasmid amino acid sequence would be changed upon clonning of my insert in. I'm wondering if this frameshift of the vector dose matter or not?
I'm very new to this, hope someone could help.
If your inserted gene has a frameshift and you want to express the gene, then yes it does matter - your gene will not have the correct protein sequence and so any function (if you get any) will not be representative of the normal gene.
Nothing has changed in the vector, you just did not read the map correctly so now your gene is not in frame. You need to redesign everything and start over.