I agree with most of the answers, but I´m facing a similar problem.
I have a band (with the expected size) on negative control and I doubt that is contamination, because in the same set of samples.
I have no bands in some of them, so how it would be contamination of water or reagents?
Unfortunately your negative control was indeed contaminated. Perhaps you can travel to a lab down the hall to setup your reactions. Frequently, if you are amplifying a target that is frequently amplified and analyzed in the same room, or a clone of which is used in your lab, you may find it gets into your pipettes, tips, solutions, everywhere. Once your pipettes are contaminated you will find it almost impossible to get rid of it. For this reason, when doing qPCR I always do my setups in an entirely different lab.
1. It could be adding a wrong sample (not water) into the negative control by mistake.
2. It could be forgetting to change the pipette tip between preparation of your sample/positive control reaction and negative control reaction due to distraction
3. It could be that the positive control and the negative control tubes were accidentally switched when adding the PCR products on the gel.
4. It could be that the positive control PCR product spilled (too much PCR product was loaded into a small well) into the well of negative control PCR product, if they are arranged to run side by side next to each other on the gel
5. It could be that when pull out the comb, the wall between two wells crack, and the positive control product leaked into the neighboring well where the negative control sample was loaded
6. It could be that your pipettes are contaminated during pipetting after repeat use.
7. (although it seems very unlikely) but I usually place my negative control tube far away from the positive control tube, avoid standing next to each other, in the heating block of a PCR machine if the space allowed
8. An interesting article just published about this issue: "A gel electrophoresis loading system to prevent laboratory contamination by amplification products (2014)". See attached paper.
i agree with Yau, it may be one of the reasons cited below, but i gess that your pipettes is contaminated, things may be explained by the absence of bands in some samples
I agree with previous answers to this. Perhaps the solution to it is to repeat the procedure and make sure that the loadings are done properly (avoiding possible switching of samples with the negative control) and importantly prevent contamination.
Best of luck.
thank you
i appreciate all people who answer me
i use your advice
but i can not understand , if idid not make a mistake in adding sample or loading in well and if as you said we have contamination why i did not see any band in one of my sample?
Unfortunately your negative control was indeed contaminated. Perhaps you can travel to a lab down the hall to setup your reactions. Frequently, if you are amplifying a target that is frequently amplified and analyzed in the same room, or a clone of which is used in your lab, you may find it gets into your pipettes, tips, solutions, everywhere. Once your pipettes are contaminated you will find it almost impossible to get rid of it. For this reason, when doing qPCR I always do my setups in an entirely different lab.
i know but if i have contamination why one of my sample does not have any bond ?
in my opinion if there is a contamination i should see the band in all samples but i see band s in all samples except one of them and see the band in negative control.
So are they all contaminated but one has an essential reagent omitted?
What is the order of reagent addition or do you prepare a mix with everything but DNA and add that last directly to each appropriate tube? (therefore likely no "no primer" control) Are you using strip tubes or singles?
Just some ideas...
I find sometimes that my samples or controls get contaminated without proper cleaning of my PCR set-up area. I always make sure to saturate my pipettors, racks, and anything that will be coming in contact with my samples, reagents, and controls in EliminASE to get rid of any possible source of contamination along with using sterilized cell culture water. By doing that my runs tend to be 99% free of any kind of contamination.
If a band is showing up in your negative control it's highly possible that either yourself or someone else may have contaminated it with a positive sample inadvertently. That can happen from time to time especially with shared controls. If you have another possible negative control to use (even water can be a negative control) then you can try that out to see if it makes a difference.
I see three possibilities: contamination, spillage, and primer dimers. If your primer set has not been fully validated, it may be forming primer dimers and give a band in the negative control. You can sequence the product in the negative control and see what it is, or try to develop new primers against your target and test the new primers with the positive and negative controls.
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