The first step in subcloning is to grow up enough material. This means either growing up a sufficient number of E. coli cells carrying the plasmid or the artificial chromosome, or infecting an E. coli culture with a phage clone. The purified recombinant vector DNA is then digested with the appropriate restriction enzyme.In some older vectors, where the insertion site is a single restriction enzyme recognition site, the insert will be flanked by sites recognized by that enzyme. The choice is then obvious. However, most commonly used vectors now have multiple cloning sites, allowing you a lot more flexibility. In particular, if it is important to be able to excise the insert in one piece, then you will need to be able to use an enzyme that does not cut the insert itself. For this purpose, you would be likely to choose an enzyme that cuts DNA infrequently such as NotI, which you can predict is unlikely to have a recognition site within the insert.On the other hand, if you want to subclone a smaller piece of the insert, you might choose a medium-frequency cutter such as EcoRI. After digestion, you would probably separate half of the reaction mixture by agarose gel electrophoresis (Figure 8.7). This can then be blotted (see the description of Southern blotting below) and probed with the same probe you used to screen the library.
With the correct DNA fragment thus identified, you can run out another gel just like the other one, cut out the DNA fragment, purify it from the agarose, and ligate it to your plasmid vector.
Artificial chromosome vectors (BAC, PAC) have inserts measuring hundreds of kilobasepairs, making it impractical to digest and separate them. An alternative approach for these is shotgun subcloning. This method is based on random physical shearing of the recombinant vector. The resulting fragments are cloned into plasmids, transformed into bacteria, and plated.A replica membrane is then lifted from this plate, and probed with the same probe. In this way, subclones containing the desired sequence can be identified.