Recently, I have done PCR for my gene of interest. in there, I got band size of my gene of interest and also in negative control (Water). I get the same band size of my gene of interest in negative control too. Could someone suggest where have I gone wrong?
Hello Muneeswari, the band being present in your negative control indicates that contamination is present in your PCR. This could have been introduced from one of your reagents (water, primers, taq etc) or from the workspace in which the PCR is performed if you handle products and reactions in the same area.
Hello Muneeswari, the band being present in your negative control indicates that contamination is present in your PCR. This could have been introduced from one of your reagents (water, primers, taq etc) or from the workspace in which the PCR is performed if you handle products and reactions in the same area.
1 agree with Michael. TRy to seperate the extraction area than that of PCR reaction area
There is contamination in your PCR reactions as mentioned above so better try to prepare your reaction mixture in sterile conditions and used autoclaved milliq water.
Hi Surya,
Try to manage for your lab a UV-cabinet for PCR for you to work in a sterilize environment in order to avoid contamination as possible. Related to your problem, it should have been a contamination in one of your reagents. I would advise you to test it by first replacing the water used by a new one and then if the problem persist you keep testing your other reagents.
Good luck!
The first thing when you want to work is to start by disinfecting your environment and working well under sterile conditions. This contamination can come from lack of biosecurity rules in the laboratory. Maneuvering must be done in the standards and well prepare the reagents and also pay attention to the water you use. I have already had this case also by the contamination of the water.
Which type of PCR do you used ?
Which gene or primers do you amplyfied ? Size of your band ?
Pls clearify, to be undarstand before answeer
Hi Muneeswari Alias Surya,
1. Be sure that there is no cross-contamination between wells while loading your samples and at the onset of electrophoresis. (In addition, you can consider having an empty well between the control and the test samples to completely rule out cross-contamination).
2. As it is most likely a result of contamination, follow the suggestions already provided by others with regards to sterilization, or using a new set of reagents where possible.
Hello
How to interpret PCR results????? The question asked is not clear because there are several types of several, which one you are interested in? But in a few words, here is my response to the amplification of DNA in the interpretation: first there will be agarose gel electrophoresis and visualization by staining of the target DNA. Finally, we will find the presence or absence of a band of the same molecular weight as the target DNA. However It is useful to know the limitations of PCR analyzes in order to correctly interpret the results obtained. Interpretation depends on the reliability of the tests used, the pathogens sought, the site of infection, and the clinical presentation. Thank
Hello I do not know if I understood our friend Elena who asks for work under the UV rays to avoid DNA contamination, but I do it before starting my work. The use of UV is very important to sterilize the work environment and some materials before work. I also disinfects the bench before I start and I'm sure I will not have any infections. Properly prepare the reagents in accordance with the biosafety rules in the laboratory and protect the biomol water to avoid contaminations without forgetting the manipulations that must be done properly. Thank you
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