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short ssDNA cloning

short ssDNA cloning - Protocols and Methods Forum

short ssDNA cloning - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 03-02-2004, 09:35 AM
Domo
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Default short ssDNA cloning



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 would like to obtain DNA sequence from cap site RNA, since not all
RNAs have polyA.

Any suggestions?

Appreciate your help,

Domo
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  #2  
Old 03-02-2004, 03:12 PM
Michael L. Sullivan
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Default short ssDNA cloning

>Hallo,

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
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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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