I tried to mutate a tyrosine residue to alanine residue for a protein gene in a pET24 vector. The cells successfully transformed and showed the mutation, but I couldn't express the protein. When I check the whole gene sequence I realized that the gene was truncated few bases pairs downstream the mutation. How could this happen? What can I do to solve this?
possibly you have introduced a 1 or 2 base insertion/deletion and created a stop codon..It may be worth checking the sequence of the mutation primer against the template in case an extra base has crept in
Also, mutations do just randomly happen. Did you sequence the vector before you transformed it into your cells?
If you sequenced your vector before transforming it into cells and didn't have this issue, then you can try just sequencing the gene in a few more colonies.
If you didn't sequence before transforming, then you'll have to start over.
As for solving it, you need to redo the tyrosine to adenine change. As Paul Rutland said double-check that your primers don't change any other sites in the vector.
Good luck!
did you see any insertion or deletion from the sanger sequence? If yes, you should repeat the mutagenesis.
Paul Rutland Thank you for the reply. But I check the size of the gene through colony PCR by using T7 and T7 terminal primers flanking the gene. So a possible erroneous stop codon insertion shouldnt show up as shorter gene length on the agarose gel. It seems gene itself got shortened. I am trying using longer extension time, maybe that helps.
If you know the extent of the short sequence then does screening clones with plasmid into deleted sequence primers help as a means of getting the full length clones....possibly mixing 5 clones at a time if there are not many full length clones and re plating any positives?
I agree with colleagues.
You'd better focus on the methods:
Direct gene deletion and site-specific mutagenesis by PCR and one reusable marker
Direct gene deletion and site-specific mutagenesis by PCR and one reusable marker using long homologous regions.
Regards, Sergey
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