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weak RT-PCR product - (Aug/17/2007 )

Hi all, I've been doing one-step RT-PCR to clone five genes from 1st strand cDNA; the same process worked very well for one gene (I've cloned it succesfully); but I'm getting weak PCR product and lots of primer dimer for the 2nd gene; I used this pcr product to do TOPO-TA cloning; I get mostly blue colonies in selection plate, there are some white colonies but when I pcr-screen them for insertion- I don't get product;

I want to get stronger band of my GOI and no or less primer-dimer in RT-PCR; can any one help me out with suggestions if any? Thanks in advance.


Did you try to optimize your PCR reaction (temperatures, concentrations etc.)?


If your primer are not good designed, If you can design new one.
If you can’t, try purifying your RT-PCR product and trying to reamplify, it has worked for me.

-aztecan princess-