Richard R. Hardy
hardy at mighty.fccc.edu
Thu Mar 30 12:15:45 EST 1995
In article <anne.2.003DC9F8 at lmb1.rug.ac.be>, anne at lmb1.rug.ac.be wrote:
> 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
> 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?
We do DDRT using the MJ cycler (PTC-100) and it generates pretty
reproducible bands. I'm not sure whether this is really your problem
> Second, does anyone know any tips or supplementary steps added to this
> protocol to make the PCR more reproducible?
There's been discussion of this topic on the net and you can search the
bionet.molbio database at the IUBIO Gopher to look over what has been
said. It's important that your RNA is high quality and DNA-free (we DNAse
treat always). We also run duplicate or triplicate samples of same cell
type in adjacent lanes to determine the variability from prep-to-prep or
PCR-to-PCR. Even with all this, we get fragments that appear differential
using DDRT that either never give a signal by Northern or else appear not
very differential. But when it works...
Member, Institute for Cancer Research,
Fox Chase Cancer Center, Philadelphia, PA
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