Using a big primer in PCR - (Nov/02/2004 )
I need to do PCR using a big primer(100bp) and the PCR product is about 500bp. What shall I pay attention to in the protocol?
The major problem with a 100 nucleotide primer is that it is probably going to form a structure. Plug the sequence into the Oligo Analyzer program at the IDT website www.idtdna.com and check for hairpins. Also check for homodimers that may result in primer dimers.
Thank you for your advice. If I have to use the primer,no other option, what shall I do to minimize the problems? Shall I do anything on the annealing time and temperature?
Thank you !
possibly add DMSO, betaine etc to stabalize the reaction more
I think you could do a Tm gradient to find the best conditions. Other option is the DMSO. You can start with a curve of concentration of DMSO, from 1 to 10% for example, and choose the best concentration for your reaction, but you must consider that DMSO can affect the accuracy of the polymerase. If you will cloning you PCR product, DMSO is not a good option.