differential display

[anne at lmb1.rug.ac.be]

Since 4 months I started up the technique of differential display at our 
laboratory using the genhunter kit. Running the sequencing gels of the PCR 
mixes went rather smootly (I thought), just following the genhunter protocol. 
After doing PCR with all the primer combinations I found only 17 
differentially expressed bands, all the other combinations gave identical 
bands. But repeating the PCR under the same conditions none of them were 
reproducible, and even on Northern blots none of these bands seemed to be 
really differentially expressed.

So I wonder what I can do to make the PCR more reproducible. 
First, we want to buy another more reliable thermocycler. The used cycler was 
the thermal cycler PHC-3 from Techne.  Now we are not knowing what to decide : 
to buy a Peltier Thermal Cycler PT-200 from MJ Research or go for the Perkin 
Elmer 2400. Has anyone good and reproducible!!! results with any of these 
cyclers? Or can anyone give me any information about influence of different 
kinds of thermocyclers and DD?

Second, does anyone know any tips or supplementary steps added to this 
protocol to make the PCR more reproducible?

Desperately searching for information,

Anne Lenaerts
Laboratory of Molecular Biology
Gent, Belgium, Europe