Hello,
I would like to add a 3xHis residues to the N-terminal of a bacterial gene to clone and express, do I need to remove the starting codon from my gene of interest (GTG) in the forward primer design or keep it? which structure should it be 1 or 2?
1. (leader and RE site - ATG-3xHis-GTG from the GOI and the last part of the primer)
2. (leader and RE site- ATG-3xHis- the primer sequence that has the start codon removed from the GOI)
Thank you in advance!
I'm no expert but i would say first at the 5' end your RE-site followed by ATG - 3xHis tag followed by the first 20 nucleotides of your GOI not including the startcodon ATG. Otherwise protein synthesis would also start after the HIS-tag and you would get proteins of interest that aren't carying the HIS-tag. These unlabeled proteins could get lost in the purificationsteps. So I would say option 2
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Optics