I am trying to PCR amplify a 3.2kb region from a plasmid so that i can use gibson assembly to put it into a different one. The desired insert has 3 genes and 2 RBS sites, one of which shares some homology with one of my primers and makes great (distinct bands) PCR product of that gene (700bp) , but only very faint ones at the desired length (3.2kb). I have tried cutting out these sections and cleaning up with a gel extraction kit (QIAQuik) but am getting very low yields (4-6ng/ul). My annealing temperature is calculated at 60C, but when I ran it at that it got no bands. I ran 2 gradients at 60-70 and 70-80, and finally got bands at 78C. I re-ran a 50ul PCR at this temperature, loaded all the product into a gel, got faint bands at 3.2kb, extracted and cleaned up but still have low yield (4-6ng/ul). My extension time is 3 mins (1min/kb) and has yielded the best, but still faint results. Any suggestions or help on how to avoid these smaller products/ensure that only the desired product is amplified?
I would try to design a primer whose 3' end extends beyond the internal region of homology of your fragment. A few non-matching bases (in theory just one) should be enough to preclude elongation
Ever tried a touchdown PCR?
Helps in many cases where PCR specificity is a problem.
Hi,
first of all could you pls give a bit more details on the used reaction setups? Background for the question is that many proofreading enzymes contain a unspecfic DNA binding domain and are annealing above Primer Tm. This might explain already the higher temps. In general I would also favor to use a Touchdown PCR here and potentially do a redseign of the Primers. This perhaps helps you as this might then overcome the binding of the region in the middle.
Sven
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