I am trying to amplify via PCR the 16s rDNA sequence from endosymbiotic bacteria of insects. I tried different concentrations and dilutions of the template. Initially, it was showing good amplification in some of the dilutions in a few samples but weak in remaining samples. When I repeat the same PCR there is no amplification. I tried several different ways to amplify it but each time I'm getting a smear. I checked for PCR components and there is no problem. The DNA isolated is sheared in some of the samples. Is shearing the DNA responsible for these things?
i think you need to verify the master mix of PCR. Though you haven't mention which reagent you are using but I suggest you to calculate Mg++ amount carefully to amplify the DNA. Inappropriate amount of Mg++ sometimes causes the problem of smearing.
Template might degraded. Remove all proteins during DNA extraction. In this case, extract by mannual procedue seems better than using kit.
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