Hi there,

If you are cloning NdeI BamHI in pET29c then His tag coding sequence is far downstream BamHI which means that if you intend to use it the protein will be far longer than the wt (at least 20aa more) and that you will have to be careful when designing the reverse primer in order to clone the gene in the same reading frame than the Histag (in fact respecting the frame of the BamHI sequence). So I would give you the advice to include both histag coding sequence and stop codon right dowstream of the last codon of the gene and right upstream the BamHI sequence so that no further aa other than the 6his will be added to the wt sequence.

Hello,

If your cloning vector already has a his tag then you do not need to add his- tag to your primers. If you are cloning your gene of interest inside the multiple cloning site, you just require to design the primers with respect to your gene. During ligation, once your insert gets ligated to the vector, the his tag present in the vector is the "tag" that you want for your protein. While designing primers, just keep three things in mind:

1. The restriction site you chose should not cleave your his- tag.

2. Do not add stop codon to your reverse primer since it is a C-terminal His tag and not an N-terminal one.

3. Make sure there is a stop codon after your His tag so that your protein expresses till the tag and do not include any unnecessary amino acids thereafter.

Good luck :)

Thank you Dominique and Dista for your advice.

So, if I understand correctly, according to Dominique, i can include a 6xHis-tag in my reverse primer? and ignoring the vector tag?

I did design my reverse primer as below, please let me know if it is correct.

5' -XXXX-BamHI-TTA-His tag(ATGGTGATGGTGATGGTG)-my reverse primer sequence -3'

Secondly, do I need to include some glycine residues between my his tag and stop codon and reverse sequences?

Many thanks,

mah

Hi again,

your sequence design is OK. I have never included spacer sequence between gene and histag. But it may be an issue for some rare cases.

Thank you very much Dominique! Very helpful.