oligo cloning - (Feb/06/2001 )
Hi out there!
Has anybody tried to clone in a 105-mer synthetic oligo (PAGE purified) into a vector. I have restriction sites on the ends and phosphorylated before annealing (95°C for 5min then unplugging the heating block). I got no positive clones. Is the oligo too long...
I presume you annealed the oligos, digest, then ligate. But why not just do a straight blunt-end ligation, then do digestion later. In this way you can check (by sequencing) whether everything is correct before you digest and ligate.
This blunt cloning method would be a good thing to try. Just be wary of one thing- Use only the bare minimum of the insert (annealed oligos) for the blunt ligation. If you have and excess of that small of an insert, it is much easier for two insert ends to find each other than it is for an insert end to find a vector end. Thus, you will end up with vectors with more than one insert in them. You can tell that this happened by doing PCR- You will see a laddering effect on the gel, dependent on the orientation that the multimeric inserts took.