Nowadays, there are many kinds of restriction sites in plasmid vectors, so it is usually possible to find a vector with restriction sites that is exactly the same as the exogenous DNA fragment itself. This has an incomparable advantage. It should also be able to digest the recombinant plasmid with the corresponding restriction enzyme to recover the exogenous DNA. Another option is to insert the fragment into any position in the vector that can produce a matching end. For example, restriction enzymes BamHl and BglII that recognize different hexanucleotides produce restriction fragments with the same overhangs, so that the exogenous DNA fragments prepared by digestion with BglII can be cloned into a plasmid digested with BanHl. This usually prevents the conjugation sequence from being cleaved by any enzymes that have been used for exogenous DNA or vector preparation. In many cases, however, digestion with restriction enzymes flanking the polyclonal sequence can extract the fragments from the recombinant plasmid. Occasionally, between the restriction sites of the plasmid and the exogenous DNA, it is impossible to find a "matched relationship". At this time, the following two solutions can be used:
1) The end of the linear plasmid and (or) the end of the exogenous DNA fragment is connected to a linker or adaptor.
2) Under controlled reaction conditions, the DNA fragment with 3 concave ends was partially filled in with E. coli DNA polymerase I Klenow fragment. As discussed in Chapter 9, this often allows those unmatched restriction sites to be converted into complementary ends, thereby facilitating the connection of the vector to the exogenous DNA. Because the partial fill-in reaction eliminates the ability of the same molecule to pair with each other, the chance of cyclization and self-oligomerization during the ligation reaction is also reduced (Hung and Wensink, 1984; Zabarovsky and Allikmets, 1986).
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