Hi,
I've been trying to amplify up a particular gene which is already in a plasmid using pcr. I have designed 2 primers for the forward and reverse directions, both of which contain slight mutations, however I couldn't get them to work when together. Because this gene came from cDNA originally, I already had primers which I knew would work when I amplified the gene straight from this cDNA for insertion into the plasmid. I tried each of the forward and the reverse primers individually with the original cDNA primer matching the R/F together and in both cases a product of the correct size was amplified up. When I used the same conditions they wouldn't work together even though the amplify separately. I have checked for primer dimers and there do not appear to be any on the gel or when checked in a couple of online tools.
Any ideas would be appreciated!
Is the Annealing Temperature the same for each of the primers? This would be the most obvious problem. Put your primers through this: https://www.thermofisher.com/ca/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html
Are the Tm times around the same value? If not, you are going to have to re-design one of the primers, so they are around the same value.
Hi Adam,
They are both around the same temperature, also they both work seperatley at the same temperature when I perform the PCR's with the original cDNA primers just not when I put them together.
Hi Connor
Cross Dimers are the only reasonable explanation that i can thought, as they work separately. If they are small enough, could be invisible in the gel. Watch 3' end homology. You can try to add enhancers (Betaine or DMSO) or touch-down PCR, i think it would be enough, taking into account that the primers work well separately....
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