Hi all,
What would be the best choice if i want to construct plasmid with only extracellular domain of a receptor? Is there any rules as i want to avoid transmembrane region..
Hi Tang,
in general, I wold just amplify the region coding the ectodomain in a PCR using appropriate primers - the latter should be carefully designed:
1) they should anneal to the template DNA on the sites flanking only the region coding ectodomain and excluding the transmemrane domain (depending wether it is on N- or C- terminal region)
2) they should have 5'-overhangs containing appropriate restriction sites (depending on the cloning site on your plasmid)
3) the most important: after designing the primers double check that reading frame of your PCR amplicon is not disturbed after cloning it into your vector
4) polymerase in PCR reaction: if your region encoding the ectodomain is relatively long (> 500 bp) I would recommend to use some high-fidelity polymerase (e.g., Phusion, Q5) to avoid point mutations
I hope I answered onto some of your issues. I can share some additional experiences if you could be more specific.
Good luck!
Peter
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