How to ligate ds-oligos into a vector? - (Aug/25/2004 )

Hi,guys:
I try to ligase a ~40bps DNA into a single digested Vector.I got this ~40 bps DNA from annealing two complementary oligos, there are two same restriction enzyme sites in both sides of oligos. I didn't phosphorylate my oligos and also didn't dephosphorylate my vector.I use the big molar ration of insert DNA to digested Vector,try to kill reaction. But I didn't get any ligation product, only this digested vector ligase itself. some person has such experience not to phosphorylate and dephosphorylate vector,and got product,but I didn't succeed. do you have some suggestions? or I must do phorsphorylation and diphosphorylation?
appreciate your help!
Viki

hi,
if i was u, i'll do phorsphorylation and diphosphorylation.
it's easy, reliable, and always save ur time and pain.
cheers!
lyre

definitley do dephosphoylation of your vektor or you'll be screenin' colonies for the rest of your life..

greetings!

definitley do dephosphoylation of your vektor or you'll be screenin' colonies for the rest of your life..   

greetings!

yes, you definitely need to phoshorylate your oligos.

PNK + ATP will do the job.

But, if you can afford it, its more reliable to order your oligos phophorylated already.

mike

If you are still having trouble, gel purify your oligos first on a polyacrylamide gel, then anneal the two oligos and phosphorylate them. Dephosphorylate your digested vector and gel extract that as well. Then set up your ligation in 10uL. I've done this many times and it will even work with the quick ligation kits. Good luck.

When ordering PCR primers or sequencing primers, typically around 20bases, it is not necessary to gel extract. When longer oligos are ordered for cloning, it is very important to have the exact sequence ordered. Since no companies can guarantee 100% efficiency in the synthesis, there will be a certain percentage of the Oligos received that is not full-lenght product. This amount increases exponentially as the oligo length increases. Gel extracting the oligos reduces the amount of shorter oligo product in your sample and thus increases the chances for a successful ligation.
I have worked with 60-90 base oligos and I always see a large amount of shorter products. If I do not gel extract first, I have never been able to clone the annealed oligos.
Good luck!

One potential problem with annealing two primers that contain restriction sites is that they also tend to ligate to themselves due to the inverted repeat nature of many restriction recognition sites.

Plug the sequences into the OligoAnalyzer program at the www.idtdna.com site and check for hairpins, homo-dimers and hetero-dimers.

For a 40 bp insert, phosphorylated insert:dephosphorylated vector ratios of 10:1 and 15:1 should be sufficient.