NEETZoologyBiotechnology - Principles and Processes
A researcher inserts a foreign DNA fragment into the Sal I restriction site of the pBR322 vector and transforms E. coli cells. To specifically select the recombinants from the non-recombinants, which of the following two-step plating sequences should be followed?
Options
- AFirst plate the cells on a tetracycline-containing medium, then replica plate the surviving colonies onto an a
- BFirst plate the cells on a non-selective medium, then replica plate the colonies onto an ampicillin-containing
- CFirst plate the cells on an ampicillin-containing medium, then replica plate the surviving colonies onto a tet
- DFirst plate the cells on a tetracycline-containing medium, then replica plate the surviving colonies onto a ch
Correct answer
C. First plate the cells on an ampicillin-containing medium, then replica plate the surviving colonies onto a tet
Step-by-step solution
The Sal I restriction site is located within the tet^R (tetracycline resistance) gene of the pBR322 vector. Insertion of foreign DNA at this site causes insertional inactivation of the tet^R gene. Thus, the recombinant plasmids will confer resistance to ampicillin but not to tetracycline. Non-recombinant plasmids will confer resistance to both. To select recombinants, the transformed cells are first plated on an ampicillin-containing medium. This selects all transformants (both recombinants and non-recombinants) an