Move to another researchers work area and set up a run using theirpipettes ,plasticware,water and pcr reagents where possible. While doing this dissemble your pipettes and clean with soapy water.Ethanol does not destroy dna. Use a fresh lab coat,Sometimes amplimer transfers from electrophoresis areas back to your pcr setup area. If all else fails then design one primer of the contaminated set to be outside the size of the contamination then the contamination cannot amplify

Have you setup a NTC reaction? That could tell a lot, I have seen NGS results get contaminated with PCR amplicons of another test. I remember when a worked one of my old jobs, one of our customers had RUNX1-RUNX1T1 amplicons contaminating the NGS data. Turns out they were also using a CE-IVD PCR kit to do RUNX1-RUNX1T1 testing with real time PCR. PCR amplicons can be a nasty persistent problem of the modern laboratory.

I recommend verifying these steps:

· Cross-contamination: Take care to prevent cross-contamination between samples by using separate equipment and workspace for the negative control. Double-check that pipettes and consumables are not shared.

· Contaminated reagents: To minimize the risk of contamination, handle reagents with caution. Use fresh reagent stocks from a different batch, store them properly, and ensure they are not compromised.

· Aerosol contamination: Employ filtered pipette tips, work in a clean environment, and keep PCR tubes or plates covered while performing actions like opening tubes, vertexing, or pipetting to minimize the potential for airborne DNA fragment contamination.

· Carryover contamination: Thoroughly clean and decontaminate laboratory equipment, including pipettes, tubes, and the thermal cycler, to eliminate any residual DNA from previous experiments that could contribute to carryover contamination.

My suggestions:

1. Repeat the experiment using new reagents, preferably from a different batch, to eliminate the possibility of contaminated reagents.

2. Set up a fresh negative control in a separate area using different pipettes and consumables to reduce the risk of cross-contamination.

3. Conduct additional negative controls using sterile water instead of template DNA to determine if the issue persists. This will help identify if the contamination originates from the reagents or the laboratory environment.

4. Ensure that PCR tubes or plates are properly sealed to prevent aerosol contamination during the experiment.

Thank you Paul Rutland for your answer but I couldn't get your last point. What do you mean by designing one primer of the contaminated set to avoid amplification? Does it mean that I have to add either a forward or reverse primer in the NTC reaction?

Thank you Muhammad Rehman Shirzad for your answer. I have a confusion regarding 3rd point from your suggestions. Do you mean that I have to add only DNase free water in the NTC reaction because normally NTC contains all PCR reagents except template DNA.

Kindly elaborate.

Thanks

What I mean about a new primer is having one primer outside of your contamination so that it cannot bind and amplify the contamination. So if we have a dna sequence represented by 1-500 bases and your present amplification is bases 200-400 with primers 200-220 and 400-380 I am suggesting design a primer to replace your forward primer of ( for instance) 160-180 then your new amplimer is 240 bases but your contamination from the original amplifications cannot work and your results will be clean again

Saiqa Aslam I mean UltraPure DNase/RNase-Free Distilled Water

Thank you Paul Rutland for your valuable suggestion but I guess it was not a contamination issue. I performed gradient PCR at different temperatures than my original temperature and now I am not getting any amplification in my negative control.

excellent news Saiqa Aslam . Well done.