generating small DNA fragments with stick ends by annealing oligos? - (Mar/01/2012 )
I need to clone a very small DNA fragment encoding a small peptide (~35bp) into a vector with a fusion protein tag. I'm thinking since this is such a small fragment, I can design two oligos such that they will anneal and the sticky ends will be as desired. I can then clone the hybridised DNA into the vector. That way I can do away with digestion with restriction enzymes and avoid difficulties in purification since the lowest cutoff for spin columns is 40bp. Does this make sense? Has anyone tried this?
Yes, that will work. People will tell you to phosphorylate the 5' ends of the oligos, but that's not necessary. Use different/incompatible ends for this, otherwise you'll have many repeats. The cutoff for spin coumns is 70bp, if you want to go lower, you can use QIAEXII or Etachinmate.