primer extension problems

[helix.nih.gov]

Hi Rolph,

It sounds as if you're having signal to noise problems . . Have you tried
using poly A+ RNA instead of total RNA? The other approach you might think
about is 5' RACE. We have had good success using Clontech's Marathon-ready
cDNA and the RACE protocols contained therein. It's a little pricey, but so
is failure. Best of luck to you!

In article <4dg0up$l20 at info.uci.kun.nl>, Rolph Pfundt
<R.Pfundt at derma.azn.nl> wrote:

> primer extension problems
> 
> 
> 
> Author: Rolph Pfundt
> 
> Email: R.Pfundt at derma.azn.nl
> 
> Date: 1/15/96
> 
> 
> 
>      I have been trying to map the 5'-transcription startsite
> 
>      of a gene. I have found a startsite using S1-nuclease
> 
>      mapping. Because this site was at a location we did not
> 
>      expect I want to reconfirm it using another technique,
> 
>      "primer extension. I have made several attempts
> 
>      to get a signal using theis technique. In the beginning
> 
>      I used a "normal" reverse transcriptase and an end-labeled
> 
>      primer of 40 nucleotides with no results. We suspected
> 
>      that the mRNA of interest had secondary structures 5'.
> 
>      To eliminate that problem we are currently using a
> 
>      thermostable RT that elongates at 70 degrees centigrade
> 
>      and chose two different primers (25 bp) that should
> 
>      confirm eachothers extension product. With the attempts
> 
>      using these primers with end-labeling we had no results.
> 
>      In the last two attempts we used these primers in a
> 
>      reaction with continuous labeling. After gel-electroforesis
> 
>      we could detect two bands. The signal of these bands was
> 
>      very very faint, and the background in the lanes was very
> 
>      high. I would like to receive suggestion how to optimize
> 
>      this technique.
> 
> 
> 
>      Thanks!!!
> 
> 
> 
>      Rolph Pfundt (R.Pfundt at derma.azn.nl)
> 
>      Department of Dermatology
> 
>      University Hospital of Nijmegen
> 
>      The Netherlands