2 replies to this topic

[seqgirl]

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!!

Edited by seqgirl, 15 December 2004 - 03:28 PM.

[tfitzwater]

Linearizing the plasmid first may increase the PCR yield.

[littlecell]

i think your dNTP concentration was too high. too much dNTP would chelate Mg2+.