Hi! I am using plant samples taken at different time points after stress to check the response of stress responsive genes but i am shocked that the band intensity of those targets is always same under stress and control...despite the fact that markers are highly stress responsive...can any one guide me about the possible problem in my pcr?
Have you tested to make sure your PCR conditions are (semi) quantitative?
Remember, PCR reactions do eventually plateau when reactants (primers, dNTPs) are exhausted, so if your conditions are sufficient to allow this, you'll never see any differences.
For example, if you're using 35 cycles of PCR, a sample with three transcripts of your gene of interest, and a sample with 300000 transcripts of your GOI will appear near-enough identical. The latter may have reached saturation at cycle 20 while the former reached saturation at cycle 34, but if you're only looking after cycle 35, you'll never know this.
Ideally you should be using qPCR for this (since it's actually quantitative) but if you're keen to do semi quantitative PCR via band intensity after gel electrophoresis, then I'd recommend setting up a large number of replicate samples (control and test) and removing one tube of each after each completed cycle beyond about cycle 20 (so pause PCR machine, remove one control and one test sample, close machine, unpause PCR).
This way you should be able to determine more accurately which cycle numbers allow you to detect an intensity difference between your samples.
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