TROPLIST: PCR on fixed embryos

[Gerhart Ryffel]

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.shtm
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