Answer:
All of them can be necessary.
Explanation:
In typical DNA cloning, the gene of interest is inserted into a plasmid, this is achieved by using enzymes that "cut and paste" DNA producing a recombinant DNA, considering this we will first need a DNA fragment to be cloned. To "cut and paste" these fragments of DNA we will need restriction enzymes (to cut) and DNA ligase (to paste), this enzyme will recognize the specific target sequence and I'll cut it, another restriction enzyme will also cut the plasmid, then DNA ligase will link the plasmid and target gene together. Now we need to introduce the plasmid into bacteria, to extract it we use glucose as a buffer to maintain the pH-controlled for the plasmid to be stable, so that linear dsDNA (sheared chromosomal DNA) is denatured but closed-circular DNA (plasmid) is not. Once we have our plasmid isolated we can put it into our bacteria (this is called transformation), this is achieved by giving the bacteria a shock that encourages them to take foreign DNA, calcium chloride can improve the results by binding plasmids to lipopolysaccharides in the bacteria. After this shock, some bacteria will accept the plasmid but a portion won't, this is why plasmids typically contain antibiotic resistance genes to allow the bacteria that contain the plasmids to survive after the application of such antibiotic, this means ampicillin is also necessary to isolate our bacteria with recombinant DNA. Finally, you can use these bacteria as "factories" to produce proteins and then obtain them by splitting the bacteria, to achieve this splitting we can use proteases, for example, chymotrypsin. NOw you'll need to purify the proteins you extract one method to do it is using the starch binding domain (SBD) that can be found in some amylolytic enzymes, we can add a recombinant proteins for transferring the starch binding capacity to the target proteins, we will observe both proteins fused to the SBDtag, only the target protein will remain over the starch granules after the wash process.
I hope you find this information useful and interesting! good luck!
