Step Two: Make Recombinant Plasmids
If DNA ligase was added to the tube of cut DNA, the sugar-phosphate backbone bonds would be sealed and the two cut linear DNA molecules would become one circular recombinant plasmid. This plasmid will have an unaltered Amp antibiotic resistance gene and an inactivated LacZ gene with the insertion. The inactivation of one gene on the plasmid while leaving the other gene unchanged is a key requirement for gene cloning.
Fig. 9: Foreign DNA with gene of interest (A, black) and plasmid (B) cut with same restriction enzyme. (Image by D. Lee)
Fig. 10: Cut foreign DNA and plasmid mixed (A), ligase enzyme added (B) and recombinant plasmids made (C). (Image by D. Lee)
The formation of a recombinant plasmid is taking place in a test tube. The cutting and ligating enzymes will work if the right combination of DNA and enzymes is added even if the gene cloner cannot see what is happening inside the tube. Because the resealing relies on chemistry and the chance encounter of sticky ends, a number of different kinds of recombinant plasmids can be made. One common result is the sticky ends from the same plasmid molecule coming together. This will give the same plasmid sequence as the original and is called the nonrecombinant plasmid. Thus the test tube of the gene cloner will contain a combination of recombinant and nonrecombinant plasmids. Their next step is to introduce these plasmids into E.coli bacteria cells.