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.
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Posted 07 December 2009 - 08:08 AM
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-...o2/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....
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-...o2/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....
Edited by DRN, 07 December 2009 - 08:09 AM.
