Band in the negative control that too of the same bp of the sample is clearly an indication of contamination with the sample. Try first adding the negative control, close the tube and then add the sample tubes.

Yes, it is contamination, you need to add negative control first and close that tube.

This is clearly pcr contamination and it can be hard to get rid of. I would clean up my working area and pcr machine. Dismantle and clean all pipettes and change to new plasticware. Move temporarily to another persons working area and using their pipettes dilute new primer stocks and either borrow or make up new NTPs Taq and use their water.Run a pcr with one positive and 3 no template controls. When you can get a negative NTC then move back to your pipettes and working area. Often contamination comes from aerosol spreading when opening pcr plates or tubes or transfer of used buffer from the gel tanks on gloves to your working area. If you cannot solve this problem then redesigning one primer to be outside the amplimer that causes the contamination will always work

Hello everyone, I did everything you said and even ordered a new primer and the band is still there :)

All new reagents and water and different pipettes and prepared in a new working area ?

@paul Rutland yes :(

Has your no template control always been positive for the lower band Merve Gündoğdu ? or has it been clean in the past. Are you able to give the primer sequences and species studied ? What is the reason for the 2 different size bands?. How many cycles of pcr are you doing. What is the source of the water that you use to dilute reagents/make up pcr mix and add to the NTC sample?

First of all thank you for interesting 🙂 there is two bands because of transformation error. I saw before and know I have to get rid of. When I obtained one band, still NTC has band also :/. I am not sure what is the right explanation and term. However, I use always sterile ampoule water for dilution (injection water in the tiny glass bottle like 2 mL). @paul rutland

It still looks like contamination to me Merve Gündoğdu but many years ago when people were growing their own Taq polymerase poor purification left bacterial dna in the taq and this could amplify with the pcr primers.. In this case the extra band would always have amplified but if you had clean NTCs early on and it then appeared it still looks like contamination to me in which case redesigning the primers to amplify a larger pcr product is worth trying....primers are very cheap.

@paul rutland thank you dear professor. You are really helpful :)