Ligation Independent Cloning
...activity back to polymerase. In our case, dGTP will be used in the reaction, meaning that T4 Pol will remove...appropriate). The primer length is dependent on the T4 Pol "chew back" reaction, in which a single dNTP is included...guanosine residue and become stalled. A typical T4 Pol reaction is shown. Mix the components (add the T4... room temperature for 30 minutes. Inactivate T4 Pol by heating to 75° for 20 minutes. Volume (μl) Reagent...will interfere with the exonuclease activity of T4 Pol in the following step. This may be accomplished ...Overhangs Treat your purified PCR product with T4 Pol in the presence of dCTP (not dGTP), corresponding...ligation. Because of its dual polymerase/exonuclease functions, T4 DNA polymerase can create overhangs of varying...