Question

1.What it is the purpose of bacterial transformation in recombinant technology?

2. What are two properties of PCR which make it a valuable tool?

3. In a sentence, describe what happens in the second step of PCR?

Answer 1

1. Bacterial transformation means insertion of foreign (gene of interest) in the host organism bacteria. This gene of Interest is inserted in the vector and then it is inserted in the bacteria. Transformation may be used for two purposes. First to get more copy number of recombinant Vector which contains gene of interest. It can be done by transforming recombinant plasmid in the DH5alpha E. coli bacterial cell. And second is to produce protein from the recombinant vector containing gene of interest. This can be archived by transformation in BL21 E. Coli. strain.

2.

It is rapid, cheap and sensitive method.

It can amplify the DNA and thousands of copies will be formed from low copy number in small duration of time.

3. Second step is Anneling.

Annealing: In this step, the response temperature is brought down to 50–65 °C for 20–40 seconds, permitting annealing of the primers to every one of the single-DNA strands. Two unique primers are used in the reaction mixture. These primers are single-stranded DNA arrangements themselves, yet are a lot shorter than the length of the target DNA and complementary to the short sequence of 3' end of target DNA. It is basic to decide a legitimate temperature for the annealing step since proficiency and explicitness are emphatically influenced by the annealing temperature. This temperature must be sufficiently low to take into consideration hybridization of the primer to the DNA strand, however sufficiently high for the hybridization to be explicit, i.e.., the primer should tie just to a superbly complementary piece of the strand, and no place else. Next, the enzyme polymerase will binds to the template-primer hybrid and starts DNA formation.