ligating small oligos - how to ligate small oligos into vector (Oct/10/2005 )
i want to ligate 90 bp ds linker/adaptor to fuse two genes, right now the linker is in pUC, i can't purify it from gel, so anybody having any idea how to get it into pET in which i want to put it, how to ligate this small insert, what ratios to use.
further, i got this as ss oligos gel purified and annealed & phosphorylated them myself, then put in pUC to make identification easier, now i want to put it in pET, so i'll appreciate all suggestions about how to purify the linker once i digest pUC and how to put it in digested pET.
i've cloned two annealed oligos, phosphorylated into a vector using a 1:5 ratio. But they were phosphorylated by the manufacturer. Assuming the proportion of phosphorylated oligos is 70% in your prep, i would say a 1:8 to 1:10 ratio is recommended.
My suggestion might be crazy: you can put all the four components together and perform a ligation. (vector, geneA, gene B, phosphorylated ds oligo)