RACE-PCR

Nico Dantuma nico.dantuma at mtc.ki.se
Wed Jun 17 06:54:53 EST 1998


>Date: Wed, 17 Jun 1998 13:56:35 +0100
>To: bmrooma at WIZARD.WEIZMANN.AC.IL (Desai Rooma)
>From: Nico Dantuma <nico.dantuma at mtc.ki.se>
>Subject: Re: RACE-PCR
>Cc:
>Bcc:
>X-Attachments:
>
>Hi,
>
>Do you use the Clontech kit? I had similar problem some time ago. Try to
>do a 3' RACE with the primer that is supplied for the cDNA synthesis and a
>gene specific primer. This worked in my hands when the normal RACE did not
>work. Note that you expect more background as this part is presented in
>all templates as double stranded DNA (with other words the specificity
>solely relies on your gene specific primer). The strange thing was that
>exactly the same template gave a nice product just a week before this
>failure (exactly the same PCR). My gues was that the single stranded
>overhang to which the (normal) RACE primer anneals is unstable. I repeated
>the experiment using fresh RACE cDNA template (synthesized the template
>and do the RACE on the same day) and that resulted in the expected
>product. My advise is to stick to the RACE strategy and check this out
>first.
>
>Good Luck,
>
>Nico
>
>>I'm trying to get the 5' and 3' sequence of a gene by RACE PCR. The
>>positive control with the two gene specific primers work, but the 5' and
>>3' RACE reactions don't. I see absolutely nothing there! Can any one
>>suggest what could be the problem. Also, is there any other way of getting
>>the complete gene sequence, besides RACE-PCR and cDNA library?
>>
>>Any help will be greatly appretiated.
>>
>>Thank you,
>>Rooma.
>>
>>__________________________________________________________________________
>>
>>Rooma Desai
>>
>>Dept. of Neurobiology
>>Weizmann Inst. of Science
>>Rehovot, Israel
>>
>>office phone: 934-2855
>
>
>


______________________
Nico Dantuma
Microbiology and Tumorbiology Center
Karolinska Institute
Box 280
S-171 77 Stockholm
SWEDEN
Phone: +46 8 7286280; fax: +46 8 331399





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