Hi Shadab
if your can elaborate a little bit more about the type of cDNA synthesis you are doing and what concentrations of reagents you are using and whether you have assessed quality of RNA before initiating cDNA synthesis and if your are doing cDNA sythesis for the first time ,whether you have taken appropriate controls for assessing which step might have gone wrong ? These details and a photograph of the smear would be more informative for people on RG for getting more insights into your problems and giving you useful tips..
bye
i am using random primers. What optimum concentration of it should be used?
I also had the same conditions previously, for a long time i couldnt get a stable product. I played with the annealing temperature.. I ran a step -up PCR with a very low annealing temperature (-13 degree of melting temperature of the primer) or few cycles say, 5 cycles followed by 30 cycles at higher annealing temperature (2-4 degree lower melting temperature of primer).
you should also play with the concentration of the cDNA used for your PCR reaction.
These 2 conditions helped me to finally have a stable product at the end
Good luck
- Badges
- Science method