gradient PCR - PCR (Dec/29/2007 )
Hi....I just want to know the difference between a gradient PCR and a normal real time PCR..thanks
Gradient PCR uses multiple samples arranged across the heating block. The block is controlled to heat one side hotter than the other during the annealing phase of the PCR cycle, allowing multiple different annealing temperatures to be tried simultaneously. During other phases of the PCR cycle, the block temperature is uniform. This is helpful if you don't know the proper annealing temperature for your PCR, and want to experimentally try many different ones in a single experiment. In general, I have found that the annealing temperature is not as critical as many suggest, and that if you are having trouble with a PCR reaction, redesigning the primers should be your first thought.
thank you sooo much...
I have got primer sequences from Real time PCR data base. They are giving primer dimers. Do I have to design primers on my own instead of standardizing with primer concentration and annealing temperature? Please suggest me?
If you can redesign them, I would, since the primer dimer activity tends to be variable and will interfere with the interpretation of qPCR results. You could try a higher annealing temperature, but it probably won't help. You could look at the primers you have with the IDT tools for hairpins and primer dimer formation. http://www.idtdna.com