2 replies to this topic

[Dan Y.]

I'm attempting to clone a ~250bp region for peptide/protein expression, and I'm considering the following strategy:
1) Synthesize a series of + and - sense oligos that anneal to each other, such that they create a (nicked) dsDNA fragment encoding my peptide with unique restriction sites at each end
2) Use ligase to join the nicked dsDNA into a continuous fragment
3) Ligate into expression vector
The modular nature should allow me to make mutations in my peptide by simply swapping out a pair of oligos.  If anyone has experience with something like this method, I'd love to hear your opinions, tips, pitfalls to watch out for, etc.

[phage434]

Main pitfalls are errors in oligo manufacture and mispriming of the assembly.  If you are really do a ligation, then the oligos need to be phosphorylated, either at synthesis time, or with a kinase reaction.  You will likely have to sequence several to many of the constructs to find the correct one, and may end up having to mutate the least errorful version to be correct.  With 250 bp, things should not be too bad.  Consider also doing pcr with low equimolar amounts of each internal oligo and normal primer amounts of the oligos at each end.  You may want to consider HPLC or PAGE purification of the oligos prior to assembly, which will eliminate the common deletion errors in oligo manufacture, and get rid of capped sequences.  It's expensive unless you do it yourself.

[gleb.kudr]

Quote

You may want to consider HPLC or PAGE purification of the oligos prior to assembly, which will eliminate the common deletion errors in oligo manufacture, and get rid of capped sequences

Yes. It is mandatory for gene synthesis!