We did the polymerase chain reaction of the plant samples whose DNA we isolated. Then we loaded it on agarose gel. As a result, there was no tape on the products. We observed a very clear and bright band in negative control. The band in negative control is not the primer dimer because it is large in size. Except for dNTP, all materials used are fresh and new. What would be the reason?
A gel picture would help but one possibility is that your sample dna quality is low. What is the OD260/280 ratio?. The amplification will be sporadic and will often fail. If you have ever used these primers before then when the NTC is positive it means that at some time in the past one sample has amplified and that you have contamination with the amplimer from this sample in your pipettes,reagents or working area
I uploaded the gel image. Smear image makers are the samples with the highest concentration. Primers were just purchased and reconstituted. Bottom and top right samples are negative control.
I recommend you to dilute the sample with the highest concentration ( for example in proportion 1:10) and run your PCR again. About the negative control - change dNTP if you are sure about the rest of the reagents.
Contamination means you need to throw away ALL of your reagents: buffer, water, dNTPS, enzyme, etc.
Clean your work area really well and try again.
Good luck!
PS never load gels with the same micropipettes you use to set up PCR
It is sad when you get a band from the negative control. It probably indicates DNA contaminations exist in your reagents (perhaps the nuclease free water or dNTP was contaminated by DNA in the process of a mastermix preparation ). By the way, I don't understand the words "there was no tape on the products".
We tried new PCR. We got rid of the band in negative control. Thank you to everyone.
Sometime, it is also important to prepare the proper gel. The agarose is usually 1.5%-2% with TAE or TBE buffer (such as 0.75g agarose + 50ml agarose). Don't use water to prepare the gel! When you are trying to run the gel, please confirm using the same buffer for gel preparation and gel running.
You might have to use new set of sterile reagents to do away with the contaminations.
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