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No PCR Product


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2 replies to this topic

#1 seqgirl

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Posted 15 December 2004 - 03:27 PM

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.


#2 tfitzwater

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Posted 15 December 2004 - 04:17 PM

Linearizing the plasmid first may increase the PCR yield.

#3 littlecell

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Posted 18 December 2004 - 12:45 PM

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




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