I need your experience and knowledge.
I have worked on mouse oocyte and 2-cell embryo .I want to do gene expression by real time PCR.
Because of a few my samples(10 cells for every time), I have worked with cell direct two step qPCR kit with SYBR for single cell (Invitrogen).I extracted cDNA by this kit and ran on gel . Only Some of them were seen on gel that my colleagues told me ,It is impossible to see very low amount of cDNA on gel.
Any way,I have tried to make standard solutions from my cDNA so first , I did normal PCR by applied biosystem fast PCR kit but I didn't find any band on gel.
I did normal PCR (10 times ) and tested Tm of primers (50,55,59, 61C) ,different concentration of cDNA and primers, manual method and everything but unfortunately I didn't see any band of PCR products on gel so I couldn't make standard solutions because I am not sure to have PCR products.
You know when I measured the concentration of PCR product by spectophotometer ,
they were ok and high.
My primers are ok and were designed and validation tested by Molecular Diagnostic center.
what can I do to have PCR products for making standard solutions?
I would appreciate if you help me ,I don't really know what to do with standard solutions.