ligation and transformation problems - (wrong) oligos (Nov/04/2008 )
Thank you for reading my post.
I've been trying to insert an oligonucleotide in a vector. Unfortunately, I've designed the oliogs wrong and the sticky ends are not (exactly) the one that I need. They will basepair with the cut vector but there will be 1 not base-paired nucleotide at both the 5' and 3' end of the insert. My question is whether E. coli can repair this?
Thank you for sharing your opinion and sugestions.
I think it depends on how much of a complimentary overhang you have but I would imagine one base on each end should still be ok. Honestly, it's impossible to say for sure so you are just going to have to try and see if it works (be sure to let us know!). Just be sure to do the anneal and ligation without any shortcuts and I'd set up a couple high concentrations of insert:vector ratios. Use T4 ligase rather than quick ligase and let it sit for a few hours on your bench before transforming. Leave the rest of the ligation on your bench overnight. If you don't have any colonies, retransform the reaction that sat overnight. Good luck!
Thanks! I hope it will work ... Can you give me a clue if it works, which is the DNA repair mechanism that would have corrected the missing nucleotide? I'm reading about DNA repair now but I'm kind of lost ...
I think you should parallelly fill up and trim your vector and DNA to make blunt ends (by T4 polymerase or Klenow) and try blunt end ligation with T4 ligase. Although the efficiency is not good, hopefully you can get some colonies, in case you fail with the reparation of E. coli. I'm trying the same method (but I just got error on one end ). It could help you save your time, good luck!
that's more or less what we are trying to do now. I used Klenow to make blunt ends, then I digested the oligos again and I hope the ligation and transformation will work (I've tried to ligate them once but I made a mistake with calculations, so Í'll have to repeat it).
Have you experienced any problems due to the different buffers? I mean when you use Klenow you need one buffer, and for the ligase - another one. How do you deal with this? Thanks for your reply in advance.
Another option would be ordering pcr primers and amplification of the oligos ...