detection of transgene by PCR - (Apr/28/2005 )
I am doing a pcr on a certain transgenic crop but my problem is that i'm getting nonspecific products. And my expected amplicon is very very faint. I already did a touchdown. I also increased the DNA in the reaction (100ng). And i think the components of my pcr cocktail is ok coz i'm getting a very strong band in my plasmid(as my postive control). I want to have a distinct and strong band to validate the presence of the transgene in the crop im working on, could you please help me? Thanks in advance.
You have to optimize the PCR condition for your screening of transgenes. As I think you will be doing this repeatedly from now on, it is worth the time and effort to go through a number of parameters.
Everytime I do a new PCR reaction (new primers or new template), I optimize the Mg2+ concentration in the PCR mix first. I do a range of concentrations from 0 - 2.5mM at every 0.5mM increments.
Usually, 0 Mg2+ gives you no bands. You want to find the Mg2+ content that will give you the strongest intensity of your desired bands.
I also optimize annealing temperature to eliminate any weak non-specific bands. If you designed your primers properly (which is the first and crucial step to getting your PCR work the way you want it to), then raising the annealing temperature should eliminate some of the non-specific bands.