5' RACE

Zhonglin Chai zchai at COBRA.PATH.MONASH.EDU.AU
Tue Jul 29 00:31:14 EST 1997


How did you clean your 1st strand cDNA before tailing? It is important to
remove the digested RNA and primers before doing TdT tailing, according to
the kit instructions. I used the same kit, and always got something after
second round of PCR using a nested primer. The product in the 1st RACE PCR
is usually invisible in gel. I use GSP1 for RT, GSP2 and acchor primer for
RACE pcr. You need re-PCR using 1uL of neat or 1/20 diluted (I would do
both) RACE PCR reaction as template, and primers GSP3 and the adaptor
primer (AUAP?).
Hope this of help.

>I have been trying to amplify the 5' end of a particular cDNA using 5'
>RACE.  I've been using the GIBCO/BRL 5' RACE kit.  I know that I have
>synthesized intact cDNA, since I can easily amplify it using internal
>primers (a nice fat product easily visible on a gel).  I believe my
>TdT is OK, since I have used it to end label double-stranded DNA with
>3' protruding ends, and I can amplify a fragment from that end-labeled
>double-stranded DNA using the same anchor primer and gene-specific
>primer(s) that I have tried using with 5'RACE.  I have tried 4
>different gene-specific primers (none of which should amplify a
>product over 500 bp), as well as nested amplification, with no
>results.  I think my problem lies with the TdT step, but I can't think
>what the problem might be, since the enzyme works fine with my control
>DNA.  Has anyone else experienced this problem?  I would appreciate
>any suggestions or advice.
>
>I've also tried ligation-anchored PCR, but with no luck there either.
>Has anyone had any success with this method?
>
>Thanks,
>
>Gae Kovalick
>Department of Zoology
>Miami University
>Oxford, OH  45056
>
>kovalige at muohio.edu



ZhongLin Chai, PhD
____________________________________________
Department of Pathology and Immunology
Monash University Medical School
Alfred Hospital, Commercial Rd,
Prahran, Victoria, AUSTRALIA 3181
Telephone: (61 3) 9276 2698 (lab)
           (61 3) 9276 2696 (office)
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email:     zchai at cobra.path.monash.edu.au
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