random ligation question?? - (Nov/23/2004 )
I would like to self-ligate some different short oligonucleiotides (26 mer) randomly, then insert into pGL3-Basic vector which have luciferase report gene, so far, only 3 copys I have pick out.....
The oligonucleiotides, commercially synthesized with the same restriction enzyme site at the both end, can self-ligate into 2 or 3or 4 or more copys, and the vector was digested with the same enzyme(Bgl2).
First, the phosphorylation of oligonucleiotide were done, then Ethanol percipitation(add yeast tRNA as a carrier to improve recovery rate), then using T4 ligase to self-ligation, after that, ligate with vector digested and dephosphorylated, after all, using JM109 to perform transformation, using PCR and electrophoresis to test copy number....
So, can anyone give me some suggestions
you know that your oligo's are single straded?
You need to order two oligos complementary to each other and hybridise them to each other - then you have double stranded DNA..