Briefly describe the major steps that need to be performed in order to clone a h

Question

Briefly describe the major steps that need to be performed in order to clone a human gene into
a plasmid. Some of the steps have already been given to you. Fill in the rest of the steps.
(a). isolate all of the mRNA from human cells
(b).
(c).
(d). Digest the double-stranded cDNA with a RE that cuts on both sides of the gene
and digest a plasmid with the same restriction enzyme
(e).
(f). Add DNA ligase to fuse the gene into the plasmid
(g).
(h). Select transformed bacteria with appropriate antibiotics
(i).

Explanation / Answer

Answer:

(a). isolate all of the mRNA from human cells
(b). Synthesis of cDNA from mRNA by using reverse transcriptase enzyme
(c). Identification and isolation of desired gene from cDNA pool
(d). Digest the double-stranded cDNA with a RE that cuts on both sides of the gene
and digest a plasmid with the same restriction enzyme
(e). insertion of gene of interest in plasmid
(f). Add DNA ligase to fuse the gene into the plasmid
(g). Incorporating the plasmid in Bacterial host cell transformation
(h). Select transformed bacteria with appropriate antibiotics
(i). The colony of cells containing the desired cloned gene can be identified and isolated

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