Team:Freiburg/Protocols/Ligation

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Ligation

Classical cloning as well as Gibson Assembly require the action of ligases to covalently assemble DNA fragments. Before ligation, those fragments anneal to each other due to complementary overhangs.

Amount	Ingredient
50 ng	Backbone
3 fold molar amount	Insert
2 µl	10X T4-Ligase Buffer
1 µl	T4-Ligase
ad 20 µl	dH2O

Incubate for 30 min at RT

Calculating the amount of insert

Ligation Excel Sheet

required Volume of Insert = 3 x Volume(Bb) x length(In) x concentration(Bb) / [ length(Bb) x concentration(In) ]

In short: V(In) = 3 x V(Bb) x l(In) x c(Bb) / [ l(Bb) x c(In) ]

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