Nested PCR problems - (Feb/08/2006 )
I´m working with nested PCR to detect two regions of animal retrovirus. In same samples, I found amplification to one region but in another region I can´t found results. There is a big variation in volume of DNA necessary to amplify each region (1 ul to 10uL). I can´t understand this results.
Another question: how do I confirm sensitivity of my reaction?
Due to the fact that you're dealing with a retrovirus, is there a lot of sequence variation? The problem might be that your primers for your nested pcr aren't annealing well for the second region.
to confirm sensitivity: make a dilution series starting with a know number of DNA copies and perform the PCR reaction and see to which dilution you're able to amplify. If you're working on cellular DNA with an integrated copy of the retroviral genome, make sure that you dilute your DNA in DNA from uninfected cells. (to be sure you're not amplifying genomic DNA).