Any liquid leakage( it can be culture medium, FBS etc) into the pipette may lead contamination. Because UV light only sterilize the outer surface of pipettes and can not reach within the pipette , contamination within the pipette can not be cleaned. I can suggest you to change the pipettes with new one you are using for pcr even they are always exposed to UV.

Hi, you can try to change the pipettes and check the laminar flow of the hood

As others have suggested, pipettes might be the problem. I recommend getting a new pipette with a fresh batch of tips.

Also, a new batch of PCR tubes might solve the issue.

Please update when you solve the issue.

Considering your sayings, sources of contaminations might be from PCR tubes, Micropipettes, their tips, and possibly your working hood.

- Use fresh and chilled PCR tubes and micropipette tips.

- You may also need to change the Micropipette and template if the problem persists.

hope it works......

Thank you so much for your answers! I'll definitely use a fresh batch of tips and tubes in the future and probably replace the pipettes if needed. I really appreciate everyone's time!

I know it's human bisulfite converted DNA, but check your template DNA for inhibitors. This happens to me a lot when extracting DNA from mosquitoes because of a pigment in their eye. Also, make sure tips and hood don't have contamination. These guys have great deals on custom designed plasmids used for qPCR standardization: https://www.yourbiohelper.com/

You can autoclave pipettes. Alternately, you can wipe out pipettes with 10 fold diluted Sodium hypochlorite and then with water. Then place it under UV for 20 minutes. UV irradiation should be done everything used for PCR before starting work and after doing PCR. Using filter tips will reduce contamination chance. There is no alternative of doing every steps with care to reduce aerosol effect. Good luck!

This is a situation where samples are amplified by a very sensitive method as we use (nested PCR) and a number of target molecules vary. The first solution is to introduce a negative control or two after each sample. This can help only temporarily, until a sample with a large number of target copies is involved in the experiment. The most common source of these problems is the opening of the tubes and the spray effect, which causes amplification in the negative controls. This can be partially suppressed by reducing the sensitivity of the amplification by omitting enhancers or by reducing the number of cycles. In general, the contamination troubles can solve the implementation of the rules that are followed in forensic laboratories, where they are working with samples with extremely low concentrations of DNA. The individual operations are carried out in three different rooms (not hoods), divided according to the expected occurrence of target molecules. The first is reserved for DNA isolation, the second for pipetting PCR reactions and the third heavy load for electrophoresis and DNA analysis, where there are the most target molecules. In addition, pipettes and centrifuges are specially designed for each operation and are cleaned (pipettes are cleaned and sterilized every three months. Three months is the time when negative controls can be kept negative. Of course, we pipette everything in a laminar box, and we have everything for one use (also ice box). The spray effect can be partially eliminated also by PCR oil, which we use in the first PCR reaction.

Following these rules, we can reliably identify 1 copy of H. pylori, C. pneumoniae or M. tuberculosis in the amplification vial.

Good luck

Thank you so much for all the input. We are going to autoclave the pipettes and I'll use the UV light before starting to work in the future.

However, I was just wondering as also the Mastermix for cDNA synthesis, beside PCR/qPCR is prepared in the hood whether you see a chance that this could be the cause of the contamination, would you recommend to prepare it seperatly?

Yes, we always do practice separate hood for mastermix preparation and dispensing. Sample, Positive control and negative control should be added in other hood. We don't have contamination problem.

If we had a contamination problems, we ran PCR without any template to prove that master mix is OK. We are using special pipettes only for master mix. We are working with master mix and samples in the same hood. I repeat that major problems are samples containing too many target molecules. So, if I did understand correctly, you can simply dilute your human bisulfite converted DNA samples. Also, you can find the source of the contamination from the sequence.

yes, it is always recommended to work separately if possible.

- If we work from the same hood two different mixes at a time there are lots of chances for contamination and you will never be free of it.

- work one mix at a time and then the other next after cleaning the hood, micropipette, and their tips, alongside using fresh PCR tubes.

- this is the easiest thing we can do to identify, doing separately and witnessing if contamination is still there or not.

change your tips in every step.

use milliQ water and sterilized Or PCR water.

use negative control.

And have pipettes exclusive for PCR.

Nina E. Hi, I think you have problem in your NFW or primer got contaminated. I suggest use new NFW and new primer mix.

I faced the same problem recently. Then I changed my nuclease-free water and got good result.

So you may double-check your water as the source of your contamination.