I tried a PCR protocol using 16s-rDNA universal primers and 55 degree for annealing and I got sharp specific bands in the exact place showing by ladder. After that I used the exact protocol again but I saw non-specific band in the 800 bp place. I tried different annealing temperatures from 50 till 63 but non of them give me the specific band. I should mention that I have changed my primers, my master mix ready to use PCR, the PCR grade water, the place I do my PCR, the other temperatures except annealing and even my DNA samples ( I extract them with different ways and repeat the things I said). Now it is just the 50 degree temperature that I can observe the bands, both specific and non-specific one in the 800 bp. I tried extracting the DNA from the agarose gel but again, I could not observe any band. What should I do now?
Thanks for helping me in advance.
It depends on what your purpose is, but if you want to sequence use your original protocol at 55 and excise the band with the correct size.
Are you running a no template control amd also a positive control sample that has worked before and what are their results. Also do you have any of the old primers to run against the new primer set and have you checked that the sequence of the new primers is the same as the old primers?
As Paul Rutland mentioned, the problem could be related to the new primer specificity. You have first to check the primer sequences, then try to adjust the primer concentration (decrease to avoid nsp bands) or increase the annealing T°.
Paul Rutland Thank you very much for your answer. The sequence of the primers are exactly the same, and one of my colleagues observed a specific band from different bacteria with exactly the same protocol. Is it possible that the bacteria I am working with have some other parts or homologous similar to primer sequences?
It is entirely possible that primer pairs amplify across species especially if they were designed long ago when fewer genomes had been sequenced. Run a BLAST search with your primers and you will get a feeling for how common these primer sequences are
What about the purity of your samples? If the purity of the samples is not right then sometimes it may happen. Sometimes, the high concentration of your DNA and primer may also cause that.
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