Best Enzymes for 5' RACE? (II)

Malcolm Moos Jr. moos at helix.nih.gov
Sat Feb 4 18:55:07 EST 1995


rmauk at calvin.bio.uci.edu (Rob Mauk) wrote:
>
> Newsgroups: bionet.molbio.methds-reagnts
> Subject: Best Enzymes for 5' RACE?
> Summary: 
> Expires: 
> Sender: Rob Mauk
> Followup-To: 
> Distribution: world
> Organization: University of California, Irvine
> Keywords: RACE, reverse transcriptase
> Cc: 
> 
> 
> 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

 I can address the first two questions on the basis of considerable experience. 
We have had excellent results using Superscript II with random primers 
according to the manufacturer's protocol, except that we routinely add
5 mM methyl mercuric hydroxide to the reaction. Hazards of the amounts 
of MMH needed for this (making a 10 µL reaction 5 mM) are overstated, and
concerns regarding secondary structure are obviated. We have done much
better with at least 1 µg polyA selected RNA in the reverse transcription.
Currently, we are using 1 µL Deep Vent per 19 µL Taq and the Vent buffer
in an air thermal cycler (the buffer is supplemented with BSA); other
mixes may well be better‹we haven't checked. Good luck.





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