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Increased cloning efficiency by temperature-cycle ligation. Nucleic Acids Res

By Anders H. Lund, Mogens Duch and Finn Skou Pedersen


Molecular cloning is a crucial, and often speed-limiting, step in many standard procedures of molecular biology. Ligation of cohesive DNA ends is normally carried out at 12–16�C to ensure a good balance between enzyme activity and stability of annealed DNA overhangs. Low temperatures generally reduce ligase activity, whereas too high temperatures may reduce cloning efficiencies by melting annealed DNA overhangs and increase overall molecular motion in the ligation reaction. Several procedures have been described to increase the efficiency ligation reactions, including the addition of condensing agents as polyethylene glycol (1) or hexamminedicobalt chloride (2). These approaches induce macromolecular crowding, and thus serve to mimic higher DNA concentrations in the ligation reaction. Other approaches seek to increase the efficiency of molecular cloning procedures by omitting the ligation step by generating long single-stranded DNA overhangs that can be annealed and transformed directly into an appropriate Escherichia coli host (3). Ligation of blunt-ended DNA fragments is normally carried out at room temperature using higher concentrations of T4 DNA ligase. We have devised a simple procedure in which high enzyme activity and DNA annealing is balanced by constant temperature cycling. We find temperature-cycle ligations (TCL) may increase the efficiency of staggered cut cloning ∼4–8-fold, while the efficiency of blunt-end clonings are increased ∼4–6-fold (see Table 1). The bacterial cloning vector pBluescript II KS + was digested with AflIII and HindIII producing two cohesive end-fragments of 434 and 2526 bp respectively, or PvuII generating two blunt-end fragments of 448 and 2512 bp respectively. All enzymes were purchased from Amersham. Digested plasmid DNA was separated on 1 % low-melting agarose (NuSieve) and purified as described by Sambrook et al. (1). For each digestion a ligation master mix was prepared containing 50 mM Tris–HCl (pH 7.6)

Year: 1996
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