How to ligate ds-oligos into a vector? - (Aug/25/2004 )
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!
greetings!
thank you very much,so I also need to phosphorylate my oligos?
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.
thank you very much, why do you purify the oligos first?
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.
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!
thank you very much.
If I use polyacrylamide gel to isolate DNA,is the same protocol as isolating protein by polyacrylamide gel?