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random ligation question??


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#1 azm

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Posted 23 November 2004 - 06:47 AM

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

#2 tuckern

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Posted 25 November 2004 - 09:38 AM

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..




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