Cloning by multiple oligo ligation
Posted 02 October 2009 - 08:09 AM
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.
Posted 02 October 2009 - 02:54 PM
Posted 03 October 2009 - 03:02 AM
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!