No PCR Product - (Dec/15/2004 )
I am amplifying a 1kb region (60% GC rich) from a 12kb DNA plasmid. PCR includes .4mM primers, .2mM dNTPs, 1.2mM MgSO4, Proprietory buffer, Pol enzyme. I am putting 1-100ng of the plasmid into the reaction and see nothing on the EtBr stained agarose gel. I've used many Invitrogen and Roche enyzme kits and tried annealing temperatures from 45-60C as well as Mg titration from 1-5mM. I've tripled checked the primer sequnces. Please help!!
Linearizing the plasmid first may increase the PCR yield.
i think your dNTP concentration was too high. too much dNTP would chelate Mg2+.