Hello everyone I did the RT PCR by using cyber green as a fluorescent dye however when I run the RT PCR of my two strains of Saccharomyces cerevisiae (duplicate) then I got the result in which the RT PCR did not quantify all the samples why? it is really confusable for me. I put all the primers and cDNA in all the well of the well plate and I am sure it was correct but RT did not quantify all just quantify 1 and 2 not all? can anyone please help me to fix my problem??
How was the concentartion of your cDNA? It is possible that you need to increase the concentration? How much cycle?
If one of your primers anneals where there is a polymorphism near its 3' end then it will not amplify normal pcr or rt pcr. Try a normal pcr with a temperature gradient starting well under the calculated annealing temperature and if you get the right size product only at a low Ta then there may be a common polymorphism stopping primer binding.
I am agree with Paul. You can also run your RT-PCR product in agarose gel to check for primer dimers or any other problems.
- Badges
- Science method