Question

What chemical you use to induce cell competent for transformation? What are three important features a vector should have?  Why it is important to have the recovery process during transformation? What is the next step after recovery process?

Answer 1

1. Calcium chloride is used to make bacterial cells competent for transformation.

2. A vector should have the following three important properties:

(a) Ori (Origin of replication) for initiation of replication of the plasmid

(b) Antibiotic resistance genes/selectable marker for selection of transformants and recombinants

(c) MCS (Multiple Cloning Site) / Polylinker site containing the restriction enzyme sites for insertion of the insert DNA/gene of interest

3. Recovery is a very important step as temperature applied during heat shock causes the cells to become stressful and thus requires recovery time.

During recovery, the transformed cells also start to express the transformed genes such as the antibiotic resistant genes.

4. The next step after recovery is to plate the recovered cells on a suitable antibiotic containing plate and incubate the same at 37 degree C to select for the transformants/recombinants.

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