You have probably carried pcr contaminated buffer from your gel tanks or opened a tube roughly and spread pcr product over your working area. You need to thoroughly clean your working area and pcr block and strip down and clean the pipettes. Get rid of all plasticware and replace with new.Get new primers and dilute them with another workers TE using their pipettes and working area since yours may be contaminated. Set up a pcr with sample and at least 2 NTC samples with their reagents and your primers. If you get a clean result your primers are not contaminated and you can go back to your area and pipettes and repeat with the new reagents. If it fails clean again and try again and if you still fail to get clean amplifications you can re design primers outside of your current amplimer and then the problem will vanish but you will have to be careful not to contaminate again with the new primers. Often contamination is from aerosol formation (droplets) when carelessly opening tube of pcr product in your working area and it gets into the dust and spreads widely.

You have probably carried pcr contaminated buffer from your gel tanks or opened a tube roughly and spread pcr product over your working area. You need to thoroughly clean your working area and pcr block and strip down and clean the pipettes. Get rid of all plasticware and replace with new.Get new primers and dilute them with another workers TE using their pipettes and working area since yours may be contaminated. Set up a pcr with sample and at least 2 NTC samples with their reagents and your primers. If you get a clean result your primers are not contaminated and you can go back to your area and pipettes and repeat with the new reagents. If it fails clean again and try again and if you still fail to get clean amplifications you can re design primers outside of your current amplimer and then the problem will vanish but you will have to be careful not to contaminate again with the new primers. Often contamination is from aerosol formation (droplets) when carelessly opening tube of pcr product in your working area and it gets into the dust and spreads widely.

Hi,

This is possibly due to contamination. Clean surfaces, door handles and other potential areas suspected to be contaminated with hypo solution.

Use fresh set of primers and pcr reagents and check. Careful Handling of pcr product is very important in minimizing contamination.

Good luck.

Did you change the primer stock? If not, it can be the possible reason than your primer stock was contaminated. So, order for a new primer set.

Another thing is the way you open/close the PCR tube caps or other caps. In this case, use fresh gloves after touching any kind of sample. And after loading the master mix in the tubes, close the template blank first and place it in a separate place during template add.

Yet another problem can happen during gel electrophoresis with all previous things ok. Migration of PCR products could be another reason. So, use 3-4 template blank in a row.

That is all I can suggest you. All the best.

If the above suggestions do not work, then you may just have to design new primers that amplify a different region that has at least one primer OUTSIDE the currently amplified product.

I once flipped a 96-well plate of PCR reactions on my workspace. I could not get rid of the contamination so I designed new primers for that amplicon.

Good luck!

Paul Rutland , Katie A Burnette , Asaduzzaman Asad , and Ajith Kumar - thank you all for your suggestions.

I changed all my reagents and pipettes once again and now I'm not getting bands in no template control.