Dear Researcher,
I facing a lot of problem in PCR amplification of deletion construct of few budding yeast gene, However the The the reaction set up, annealing temp. (55 degree C) standardized earlier without any non specific bands and also able to get few true transformants. Now I am trying to amplify the same gene deletion construct but after several trial no amplification observed. When I put gradient PCR between 55 to 65 degree C, amplification observed in 62, 63 and 65 degree C with so many non specific bands. What's the reason behind this please suggest me and how can I overcome this problem.
I have used two deferent high fidelity polymerase (Phusion HF DNA pol and iProof HF DNA pol by thermo and BIO RAD respectively.
The Ta will change if salt concentrations,the polymerase, the volume and type of pcr machine, the presence of organic chemicals ,the concentartion of primer and the degree of degradation of the primer has changed, Primers may degrade in water if nucleases are present in the water so most people use TE buffer to make up and stor primers, I do not see why degraded shortened primers would anneal at a higher temperature but they could anneal at many pleces. If this result came from plated bacteria then you could get the pcr product from wetting of the gel with unincorporated construct but then on growing the cells onwards the result would be negative. I wonder if something similar is happening with your yeast
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