The typical procedure to obtain cDNA from genomic extracted DNA is the following

Question

The typical procedure to obtain cDNA from genomic extracted DNA is the following:

RNA is extracted from desired tissue, RNA/DNA hybrid is obtained by virtue of reverse transcriptase RNA dependent DNA polymerase, H RNAse creates gaps in RNA of DNA/RNA hybrid and with the aid of a typical DNA polymerase dsDNA is transcribed, right?

Then, the newly synthesized cDNA is linear (like a "small chromosome")? How is it protected from the nucleolytic activity? Is the procedure really like I described or does the reverse transcriptase already give a dsDNA from single stranded mRNA?

Explanation / Answer

Here is the rough procedure that I've used to prepare cDNA before

How is [the cDNA] protected from the nucleolytic activity?

If you are digesting the product of reverse transcription with RNAse H, that enzyme digests RNA specifically, so your DNA is unharmed. If you are going with an alkaline conditions treatment, then the RNA will be degraded in those conditions much more quickly than the DNA due to the presence of the 2' OH group in RNA which can act as a nucleophile for a self-cleavage reaction.

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