Reverse transcribing GOI mRNA to cDNA for cloning? Gene specific primer or Poly - (Jan/22/2014 )

I plan to reverse transcribe mRNA from my gene of interest into cDNA then clone it. Starting material will be total RNA from normal cells expressing my gene of interest. I am going to use a high fidelity reverse transcriptase (i.e. recombinant with proofreading activity). 

Question: For making cDNA to clone, Is it better to use gene specific primer in this case or use Poly T? I have always used PolyT/random hexamer mix because I wanted total RNA copied to cDNA but not sure in this case. Definitely do not need random hexamers. Any suggestions or comments about this? I am not sure if it really matters because either way I will end up performing PCR on the cDNA to amplify and clone it. 

Also, the ORF for this gene is 5kb. 

Thanks!

Either should work, though using gene specific would eliminate a lot of potential non-specific binding products from the PCR step.

You may find it difficult to PCR a 5 Kb cDNA in one piece. Random hexamer primers followed by cloning in 1-2 Kb pieces would be more certain of working.

phage434 on Thu Jan 23 22:15:24 2014 said:

You may find it difficult to PCR a 5 Kb cDNA in one piece. Random hexamer primers followed by cloning in 1-2 Kb pieces would be more certain of working.

What is the cause of difficulty for PCR of a 5kb cDNA? Just asking because it does not seem obvious to me. Maybe I am too trusting of the reagent package insert sometimes. I figured if the RT can make cDNA up to 10kb and has proofreading activity it should be ok. Then PCR with a high fidelity DNA polymerase capable of generating products up to 10kb. 

Cloning in 1-2kb pieces would need to be done with blunt end ligation, right?

Thanks!

RT enzymes have improved a lot in the past few years, but still are not at the level of good PCR enzymes. There is a tendency to produce less than full length fragments. It's worth a try, however, but I would order a few primers in the middle of your gene, since they are cheap and probably useful in any case for sequencing.

Blunt ligation would be one of the very last things I would use -- overlap PCR  would be a first choice.