Cloning serves two main purposes. First to allow a large number of recombinant DNA molecules to be produced from a limited amount of starting material. Second important function is purification. Ligation mixture usually contains:

1. Unligated vector molecules
2. Unligated DNA fragments
3. Vector molecules that have been recircularized without new DNA being inserted
4. Recombinant DNA molecules that carry the wrong inserted DNA fragment

Unligated vector molecules and DNA vectors are usually degraded by bacterial enzymes. Self ligated and incorrect recombinant plasmids replicate just as efficiently as desired molecules. The challenge is therefore to identify the colonies that contain the correct recombinant plasmids.

Transformation - uptake of DNA by bacterial cells

Most species of bacteria are able to take up DNA molecules from the medium they grow in. Often DNA molecules are degraded but occasionally they are able to survive and replicate in the host cell. In particular, this happens if the DNA molecule is a plasmid with an origin of replication recognized by host. Most species of bacteria are not efficient at uptaking DNA.

To induce transformation, we place the bacteria in ice cold salt solution. Then we briefly raise the temperature to 42 degrees. This cell is now called competent.

Cells that contain the plasmid pBR322 are resistant to antibiotics. This is used for selection. It allows us to distinguish between transformants and non-transformants.

Insertional Inactivation
Harder problem to solve is to determine which of the transformed colonies comprise cells that contain recombinant DNA molecules, and which contain self-ligated vector molecules. Insertional inactivation is the inactivation of a gene by inserting a fragment of DNA into the middle of its coding sequence. Any future products from the inactivated gene will not work because of the extra codes added to it. Recombinants can therefore be identified because the characteristic coded by the inactivated gene is no longer visible.

pBR322 contains genes which code for ampicillin resistance and tetracycline resistance. BamHI cuts in the middle of the gene which codes for tetracycline resistance. If a gene is inserted here, the plasmid loses it ability to code for tetracycline resistance. Thus the plasmid containing the recombinant gene is resistant to ampicillin but sensitive to tetracycline. To screen, we use replica plates.

The pUC8 plasmid is ampicillin resistant and contains a gene lac Z' which partially codes for β galactosidase. To make the plasmid capable of coding for the whole protein, we add the missing DNA along with the recombinant gene. The host which contains the plasmid pUC8 is resistant to ampicillin and is also capable of producing β galactosidase.

Eukaryotic Transfection

This equivalent to transformation, with the difference being that phage DNA rather than a plasmid is involved. Just as with a plasmid, the purified phage DNA or recombinant phage molecule is mixed with competent E. coli cells and DNA uptake is induced by heat shock. Transfection is the standard method for introducing double-stranded RF form of an M13 cloning vector into E. coli.

Prokaryotic Transduction - in vitro packaging of λ cloning vectors

Transfection of λ phages is not a very efficient system when compared to infecting the bacteria with recombinant λ phages. The idea is to first create recombinant λ phages and then to infect the bacteria with them.

Source

Gene Cloning & DNA Analysis by T. A. Brown