short ssDNA cloning

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Tue Mar 2 10:12:51 EST 2004


>Hallo,
>
>Could somebody give an advice how could I clone single stranded DNA?
>
>To conform full-lenght mRNA, I am planning to perform RACE with
>specific DNA oligo, which binds ~100 nt (50 - 200 nt ok) bp from
>5'-end of mRNA.
>Then I want to clone this short ssDNA oligo and sequence it.
>
>It is possible to introduce specific sequence to 5'-end of oligo. So I
>could capture this end using specific restriction site with dsDNA
>vector. Hovewer, the problem remains with random 3'-end. Should I use
>random adaptor NNNNN?
>

I don't think what you are describing is actually RACE, since the "A" 
in race stands for amplification.  Sounds more like what you are 
doing is primer extension.  Frankly, without a PCR amplification 
step, I think cloning of the resulting single stranded DNA product 
would be difficult.

Why don't you do a real RACE protocol, where after reverse 
transcription, you A-tail the product, then use a modified oligo dT 
primer to to make second strand.  The resulting molecule can then be 
amplified and pretty easily cloned.

There are numerous protocols out there for doing RACE, as well as 
commercially available kits (e.g. I think clontech makes one).

I hope this helps.

Mike
-- 

Michael L. Sullivan, Ph.D
Research Plant Molecular 
Geneticist                                                            
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5397 Phone
(608) 264-5147 Fax
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