Hi guys,
See attached the amplification curves which I have been getting over the last few weeks. The plates are 96-well and has 7 samples which are run in triplicate for two genes. The genes which we are looking at are ACTB and PLK1.
Anybody know what can be done to optimise this? The samples are cDNA which has been prepared from isolated PC-3 RNA. I have treated all samples with DNAse too.
Any help is greatly appreciated.
Firstly you need to ensure that empty wells are omitted from the analysis.
Part of your problem is the baseline setting, because you have a very low Ct values. You need to modify the "Analysis Settings" - change to "Manual Ct", and then change to "Manual Baseline", and click apply. You will then get two red triangles on the X-axis; these define the region used as the baseline. You need to move the right-hand triangle to below the cycle number where you begin to get amplification. This should sort out the problem with the curves that have a gap in the middle. You will also be able to move the threshold line to a suitable place to pass through the correct parts of the curves. Once you've done this you can look at the curves again and redefine any remaining problems.
in the second figure apparently there is excess DNA, diluting the sample may solve the problem.
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