Talk:DNA ligation

General ligation protocols are fine for protruding sticky end ligations but often fail with blunt ends or recessed sticky ends. For blunt ends, higher enzyme concentrations are recommended along with considerably lower total DNA concentrations (cf. Bercovich et al., BioTechniques 12(2), 190-193 (1992)). If there is someone around with a good working protocol for ligation of blunt and of protruding ends, it would be much appreciated. {MDolinar, July 25, 2007}

Insert to Vector ratios

Hi, new to this wiki but not to wiki's. I notice that a formula was used for a 6:1 insert:vector ratio at the top of the page but later it talks about the appropraite range is 1:1 to 5:1, this seems like a contradiction. Under what conditions does one use the 6:1 ratio. This page needs at least some discussion of how to ligate vectors to inserts where both are reasonably long. Phil 18:22, 6 September 2007 (EDT).

• Reshma 19:50, 6 September 2007 (EDT): Good catch. I think this may reflect the fact that depending on exactly what you are ligating, the optimal insert:vector ratio can differ so different people advocate different ratio's. Hence the note in bold regarding the insert:vector ratio. If you have suggestions on ligating long inserts, please feel free to add it to the Notes section.
  • Not suggestions but questions. I apparently have a setup that is hard to insert. V6.3kb:I2.2kb and have only the slightest evidence that V:I has formed, let alone circularized. I am noticing in the literature that optimal ligations are between 4 and 16'C (O/N) and that ligation ratios are between 1:1 for 'cip' treated vectors to 5:1 for blunt end ligations. Lots of contradictions, also people recommend vector concentrations of 10 fMole and insert of 30 fmole, but then say don't add to much DNA, no more than 20 ng per. Some people recommend preheating the vector and insert after mixing 37'C, 50'C, 65'C (drive iff residual ethanol or unstick the ends). For the first time user it is helpful to know what is most important (fmoles/ul or ngs/ul), what to do and when to do it.
    • Reshma 14:21, 7 September 2007 (EDT): Ahh ok. How is your insert generated? By PCR followed by digestion or by digesting it from another vector? If it is digested from another vector, does the insert vector and the ligation vector have the same resistance marker? Have you've tried this cloning once already and it failed? If so, can you describe your conditions?