Posted 06 April 2011 - 02:53 AM
I am very confused with my results now. I have extracted RNA from tomato, synthesized cDNA and did PCR reaction. I also ran the PCR using gDNA. I got the same size of bands for my cDNA and gDNA. Supposely, my PCR product should be around 300bp and and my gDNA (with inton) is around 500bp. All the bands that I got are around 500bp. Than I repeated again my experiment because I thought that my samples have been contaminated with gDNA and I have tried varities concentration of MgCl2 for my PCR reaction (from 1mM MgCl2 to 50mM MgCl2. After ran the gel electrophoresis, I found that MgCl2 at concentrations of 25mM to 40mM showed that bands only for my cDNA, not my DNA and not my ubiquitin (housekeeping gene). Meanwhile, at concentration of 2.5mM, I got the band for my cDNA, ubiquitin from my cDNA and DNA, but not my gDNA sample. I also have ran together my control which are:
1) gDNA + cDNA
2) using random template to synthesize cDNA
3) PCR reaction without sample, only primer and PCR kit
4) PCR reaction using RNA
All the controls showed no bands.
Can someone help me with this? I have designed the primer using coding sequence (without intron)