earn Swiss chocolate: a tough RT-PCR / RAPD nut

[VOGEL at ubaclu.unibas.ch]

Hi folks,

here's a tough nut to crack for you PCR jocks.
Since my differential displays on zebrafish RNA (which worked fine one and a 
half year ago) constantly failed when I started working on them again
recently, I started a series of experiments to find out where the 
problem is/was. (I can supply informations on the exact way the 
differential displays failed, but this is probably of no concern here.  
If you think you want to know more, mail me. But this here should be tough 
enough.)

* To check if the reverse transcription worked, I reverse transcribed 
total RNA that was either highly pure or freshly prepared by a method 
routinely used in the lab. The RT was performed with two different dNTP 
concentrations and with a T12 primer (from the diff.disp.) and a specific 
primer. 
Then, I performed PCR with two primer sets that produce either a short or 
a long (almost full cDNA length) amplificate and allow discrimination 
between RT product and genomic DNA. As a control I included genomic DNA 
(final 0.5ng/ul). All reactions worked and produced the expected 
amplificates; further, it showed that the freshly prepared RNA contained 
genomic DNA contamination.
---> specific RT-PCR works

* To try conditions closer to diff.display, I then performed PCR under 
the following conditions that are in fact those used for RAPD:
0,2uM decamer primer (from diff. disp.); 100uM dNTPs; 2,5 ul RT product 
(from the very same tubes used in the experiment above kept on ice for 
some 5 hr between experiments); 25ul total volume; 
92C/2' - 36x 92C/1', 36C/1', 72C/1' - 72C/5' (I also repeated the 
experiment with the denaturing steps as 94C/5' - 94C/1' with the same result.)
Control was again genomic DNA (final 1 ng/ul) and the internal control of 
the genomic DNA contamination.

The control reactions produced nice laddery amplificates.
The RT products produced NOTHING AT ALL, not even a smear or unspecific 
artifact bands, just plain - nothing.
---> ???

A substantial reward of Swiss nut chocolate will be given to whoever puts 
me on the right track to pin down the problem!!

Andreas

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Andreas Vogel
Biocenter of the University
CH-4056 Basel
vogel at ubaclu.unibas.ch