primer design - (Dec/07/2009 )

Dear All,

I am struggling with PCR primer design. I designed several sets of primers amply genomic DNA (actually for colony PCR). None of them worked. I was wondering which websites you are using to design primers and to test the quality of primers.

When I check Tm for my primers, different websites give me different Tm. The parameters of different websites are different. I was wondering if I need to change the values of the parameters in the websites. What salt concentration I should use when doing Tm calculation. Some websites have Na+ concentration, but some have salt concentration. When it is salt concentration. Does it mean all salts, for example, Tris, K+, Mg++ and so on?

Sorry for the stupid questions and thanks a lot for your kind reply in advance.

Hi

You can use NCBI primer design tool for designing ur primers....u may check the Tm of ur primers using the website:
http://www.dsi.univ-paris5.fr/bio2/OligoTM.html i rely on this one n it works fine for me.....
U need to take care of the GC/AT content and the length of your primers as well.......i keep the length to about 23-28 bp n the Tm to about 55-59 C..........thats coz the organism that i m working on has a AT rich genome........similarly just chk all this criteria for urself also.........hope this helps

best....