Ligation of DNA fragments by homopolymer tailing
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10 × tailing buffer (1 M sodium cacodylate, pH 7.0, 10 mM CoCl2, 1 mM DTT)
Method
1. Prepare a tailing mixture of 50 l containing DNA, 1 × tailing buffer and a 20 M concentration of the nucleotide to be added to the DNA.
Note
Choosing the appropriate complementary nucleotides to “tail” the termini of the two DNA fragments to be ligated may facilitate further subsequent analysis. If the aim is to ligate DNA fragments of interest to plasmid vectors in order to produce bacterial clones, it is sensible to design the homopolymer tailing based ligation procedure so that the subsequent analysis of cloned DNA fragments (e.g. analysis of cloned DNA fragments length) is user-friendly. For example, digestion of the vector DNA with the restriction endonuclease PstI (recognition site: CTGCAG) which will produce the 3’- extension TGCA at both termini. If the PstI digested vector DNA is then homopolymertailed with dGTP, and consequently the target DNA fragments are tailed with the complementary dCTP, then successfully ligated recombinant DNA products will now contain two PstI recognition sites either side of the target DNA. Therefore, simple digestion of the cloned recombinant DNA with PstI will liberate the cloned DNA fragments (including homopolymer tails) from the vector DNA.