If I wanted to clone a gene from scratch since I dont have a plasmid I can take the gene from, how do I do this?
From general reading, it seems people either extract DNA, carry out restriction digest and get a mix of products which contains introns and exons which I find really confusing.
Or we can carry out RNA extraction, convert to cDNA using reverse transcriptase and then design primers to gene of interest. To me the RNA method sounds simpler since there are no introns that need to be got rid of.
I dont have any protocol and wondered if anyone has a protocol they can share to isolate my gene of interest from RNA and then carry out Gateway cloning? Is the method of RNA extraction and cDNA generation for cloning different to that used for qPCR?
Any advice would be greatly appreciated.