My protein contains two equal sized domains of about 120 amino acids each. I have designed an engineered protein where one of the domains has been replaced with a small loop. I do not know how to design a vector for cloning and expression of my protein. I currently have plasmids coding for the entire two domain protein. Any advice would be appreciated! Thanks!
If you want to exchange the coding region for the 120 amino acids (360bp) of your plasmid with a coding region for 6 amino acids (18bp) you do following. You design a primer consisting of 20bp upstream of the 360bp +18bp coding for the 6 amino acids + 20bp downstream of the 360bp. This is your first primer (58bp) the second primer is the reverse complement of your first primer. Perform a PCR with both primers and your plasmid and amplify the whole plasmid (16 cycles). Digest your input plasmid with DpnI (just add 1µl to your PCR reaction and incubate over night at 37°C) and transform 2-4 µl in DH5alpha and test a few clones (either control digestion or better sequencing). Most of them should have the 360bp exchanged with the 18bp. Please let me know if anything is unclear.
http://onlinelibrary.wiley.com/doi/10.1002/prot.21786/full
See Fig 1C for deletion/substitution. In your case you can design your primers so that overlapping sequence encodes the 5 aa loop.
If you want to exchange the coding region for the 120 amino acids (360bp) of your plasmid with a coding region for 6 amino acids (18bp) you do following. You design a primer consisting of 20bp upstream of the 360bp +18bp coding for the 6 amino acids + 20bp downstream of the 360bp. This is your first primer (58bp) the second primer is the reverse complement of your first primer. Perform a PCR with both primers and your plasmid and amplify the whole plasmid (16 cycles). Digest your input plasmid with DpnI (just add 1µl to your PCR reaction and incubate over night at 37°C) and transform 2-4 µl in DH5alpha and test a few clones (either control digestion or better sequencing). Most of them should have the 360bp exchanged with the 18bp. Please let me know if anything is unclear.
Thanks for the answers! I will let you guys know how it goes!
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