bluescript and RACE

Stephen Klautky agoodrid at vaxa.weeg.uiowa.edu
Fri Jun 5 01:55:00 EST 1992


In article <9206022255.AA15232 at calvin.jci.tju.edu>
buchberg at CALVIN.JCI.TJU.EDU (Dr. Art Buchberg) writes:

> 2) WE are currently trying to amplify the 5'ends of several mRNAs
> (5'RACE) are are running into some problems.  We think the TDT rxn is
> not performing well, We are currently trying to use labelled C to check
> for incorptoration.
> WE are using the BRL kit, which uses a poly C addition.  Controls
> indicate that the RT is working well, WE can amplify using an oliog
> located in 5' region. But we want to know how much further 5' our genes
> are.  Any suggestions or commonly encountered problems with RACE would
> be appreciated

I am also interested in the 5' RACE procedure.  My protocol is similar
as that described in Frohman, PNAS 85:8998-9002.

After the 1st amplification using oligo(T) and a nested primer (relative
to one used in RT) I need to do a second amplification with a second
nested primer and oligo(T).  At this point I can detect a wide band of
products that are specific to my gene of interest based on Southern
analysis.  The oligo I use for probing is nested yet again.

I have not tried to clone DNA from the last amplification or further
confirm their existence.

The protocol and kit put out by BRL (and described in Focus 14:46-52)
looked pretty promising.  Is this the protocol you are refering to?
I was thinking of buying the kit but do not know anyone who has tried
it.  Any information someone can offer would be appreciated.

-=] Steve K. [=-       AGOODRID at VAXA.WEEG.UIOWA.EDU



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