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dna ligation protocol for cohesive end

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dna ligation protocol for cohesive end

Postby genetherapy » Thu Jan 26, 2012 7:46 pm

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

Thank you
genetherapy
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Postby JackBean » Fri Jan 27, 2012 8:23 am

you mean sticky? What the problem? Just add DNA ligase, some ATP and keep at 17°C or something like that.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby genetherapy » Fri Jan 27, 2012 3:54 pm

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
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Postby JackBean » Mon Jan 30, 2012 11:09 am

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.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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