dna ligation protocol for cohesive end

[genetherapy]

Hi,
Do anyone suggest me an efficient protocol for cohesive end ligation for cloning?

Thank you

[JackBean]

you mean sticky? What the problem? Just add DNA ligase, some ATP and keep at 17°C or something like that.

[genetherapy]

yes,sticky end ligation. In some protocol it says keep in 16 °C overnight,some says 20°C 4 hours and some says 4°C .I am mixed up the ligation ratios of insert and vector also important. In some protocols it says keep the vector and insert at 65°C to avoid selfligation and then put ligase etc. Please help me if there is an effective protocol that works well

[JackBean]

we usually use 16-17°C for O/N. The point of such low temperature is that the sticky ends are short thus the pairing is not much efficient.