TROPLIST: PCR on fixed embryos

[Takuya Nakayama]

In my experience, I could have done genomic PCR after in situ  
(including treatment with MEMFA, bleaching with H2O2 and formaide but  
NOT by UV).  I also used the same kit but WITHOUT additional boiling  
step.  Also, my PCR products were 100bp-300bp in length.

Takuya in Grainger lab


On Jan 22, 2008, at 7:53 AM, Gerhart Ryffel wrote:

> Dear colleagues,
> we try to determine by PCR transgenes in Xenopus embryos that have  
> been analyzed by whole mount in situ hybridization.
> The in situs involve MEMFA fixation as well as bleaching with H2O2,  
> formamide under UV.
> We tried the Qiagen DNeasy Blood & Tissue Kit (Cat. No 69506) and  
> used an additional boiling step (20 min. 99 degree) to reverse  
> cross links. We could never detect by PCR a transgene or the  
> endogenous ODC.
>
> May be we should leave out the bleaching or use an alternative  
> method for fixation?
>
> Thank you very much for any suggestions.
> Gerhart Ryffel
>
>
> Prof. Gerhart U. Ryffel
> Institut fuer Zellbiologie
> Universitaetsklinikum Essen
> Hufelandstrasse 55
> D-45122 Essen
> Germany
> Tel:  ++49 (0)201 723 3110   Fax: ++49 (0)201 723 5905
> http://www.uni-duisburg-essen.de/home/fb/ifz/forschung/ae/ 
> de_startseite.shtml
>

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