Plasmids contain the bacteria’s genes for antibiotic resistance.
? If a restriction enzyme is used which cuts across this gene and a DNA fragment is
inserted, it follows that the bacteria will no longer be resistant to that antibiotic.
? This can be used to identify the bacterial cells that have taken up the target gene.
1. After transformation, the cells are plated onto a sterile plate containing a
nutrient agar containing ampicillin and incubated for about 24 hours.
2. Only those cells which took up plasmids will be resistant to ampicillin and
survive to grow on as colonies.
3. A replica plate is made using a velvet cloth and the cells are transferred to a plate containing tetracycline (the antibiotic for which the target gene was inserted across its resistance gene).
4. Following incubation some colonies remain resistant and some die.
5. Those colonies which died on the second plate must therefore contain the correctly transformed bacteria which can express the target gene.
6. These colonies can then be identified on the first plate and then samples removed and cloned in a fermenter