I'm running a conventional PCR in order to detect Mycoplasma spp, however i'm having some problems as some negative samples sometimes amplified and I see the band in the gel and sometimes I do not see it. I've tried three different times and in the 1st try I got the samples as negative, but then in the 2nd and in the 4rd try I could see some band (Not as clear as in the positive control) and not with the same intensity in the duplicate. I tried the three times with different water and reagents, so my questions is: It is possible to contaminate the samples that are negative with the Mycoplasma DNA from the positive control ? or Do you have any idea about why this could be happening ?
It is possible that you negative control is contaminated. I would verify my controls before running the experimental samples. You can use a commercially available negative control in case you don't have access to one.
go back to original stock of negative control and make sure that you sterilize the tube before opening and use gloves for mycoplasma contamination usually comes from human skin.
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