Best Enzymes for 5' RACE? (II)

Rob Mauk rmauk at calvin.bio.uci.edu
Fri Feb 3 23:00:05 EST 1995


Newsgroups: bionet.molbio.methds-reagnts
Subject: Best Enzymes for 5' RACE?
Summary: 
Expires: 
Sender: Rob Mauk
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Distribution: world
Organization: University of California, Irvine
Keywords: RACE, reverse transcriptase
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I would be very interested in hearing opinions on any of the following:

   1.  What is the enzyme of choice for 1st strand cDNA synthesis for 5' 
       RACE, given that my 5' end is probably 3kb or so away, and also 
       that there is likely some nasty secondary strux to be crossed? 

   2.  Would a gene-specific primer or random hexamers be preferable?

   3.  What is the preferred combo of polymerases for long distance PCR?

I have made and screened two cDNA libraries of the same tissue, one of 
which is dT-primed, and the other random-primed.  All the clones I have 
pulled out have the same 5' end, which I am certain is not the true 5' 
end.  I plan to try the Clontech marathon cDNA RACE kit, perhaps 
substituting a thermostable reverse trancriptase.  (Anyone have 
experience with rTth?)  5% DMSO in the RT rxn has also been suggested to 
me.  Curiously, despite this kit's name, it does not come with any 
enzymes for the PCR, hence question #3 above.   Regarding first strand 
primers, would the use of random primers be compatible with elevated RT 
rxn temperature?  Is part of the logic in using random primers that they 
aid in cutting down on RNA secondary strux?  (BTW, my RNA looks fine on a 
Northern at 8kb.)  Any comments or suggestions, posted or mailed, will 
be very greatly appreciated.  I will summarize response.  Thanks in 
advance.   Rob



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