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Reverse transcribing GOI mRNA to cDNA for cloning? Gene specific primer or Poly

cDNA Reverse Transcriptase Cloning RNA Primers

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#1 Epigeneticist

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Posted 22 January 2014 - 02:56 PM

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!



#2 bob1

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Posted 23 January 2014 - 12:24 PM

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



#3 phage434

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Posted 23 January 2014 - 02:15 PM

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.



#4 Epigeneticist

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Posted 23 January 2014 - 08:46 PM

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!



#5 phage434

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Posted 24 January 2014 - 05:47 AM

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.







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