When a no template control amplifies the right size product instead of no amplification then you have post pcr contamination in some of your reagents or just contaminating your working area or pipettes.

. Replace all PCR reagents with fresh ones and wipe down the area properly and clean the pipettes inside and out as well. Try setting up just the no template control and a positive control using another workers pipettes and working area before proceeding with the test samples. additionally once you have eliminated the contamination try to keep post pcr ( opening tubes,loading gels and running gels separate from your pcr setup area. If you cannot clean up the problem ( and it will only get worse with time) then design at least one of your pcr primers so tat it is outside of the current primer then the contamination cannot amplify. Also thoroughly clean all of your pipettes inside and outside and try not to use pcr setup pipettes for preparing gel samples and loading gel samples.

Often contamination comes from gel tanks where pcr product has leaked into the buffer so use different pipettes for pcr setup and for gel loading and if you handles run gels then dispose of the gloves before going back to your work area or the wetting of the gloves will transfer dilute pcr product from the gel tank to your workbench and to everything you touch.

It will not be much pcr product but 10 cycles of pcr is 1000 times more pcr product and 20 cycles is 1000000 times more product so you will have to be very careful when opening tubes (aerosol droplets) or handling post pcr material. If all else fails then design one pcr primer outside of one of your primers ( larger pcr product) and the contamination will not amplify until you get new contamination with the new primer set

Dear

Lina Banten , Your issue likely stems from contaminationor non-specific amplification. Here are key troubleshooting steps:

1. Primer-Dimer Check – Run a no-template control (NTC) on a gelto check for primer-dimer formation. Also, analyze your melting curve for unexpected low-Tm peaks.

2. Reagent Contamination – Use fresh aliquots of water, primers, and master mix. Prepare reagents in a contamination-free area, ideally with aerosol-resistant tips.

3. Pipetting Cross-Contamination – Ensure dedicated pipettes and tipsfor pre- and post-PCR steps.

4. Annealing Temperature Adjustment – Try a gradient PCRwith temperatures from 61–65°Cto improve specificity.

5. Template Quality – Ensure DNA purity (A260/A280 ~1.8–2.0) and run a control PCR with universal plant primers to validate extraction.

6. Primer Design Issues – Check for self-complementarity or mispriming using tools like Primer-BLAST. If needed, redesign primers with higher specificity.

7. PCR Cycling Optimization – Shorten extension time and reduce primer concentration (e.g., 0.2 µMinstead of 0.4 µM).

If primer-dimers persist, consider using high-fidelity polymerases with hot-start technology to enhance specificity.

Thank youa!

As other have said, you have contamination. Remember, your goal is to eliminate the contamination so you can continue with your science. You don't need to discover the source of the contamination as long as it's gone.

Along with getting all new reagents, wiping down, cleaning pipettes, etc. I'd recommends you throw away ALL disposable items on your bench.

That includes: pipet tip boxes, tubes, gloves, kimwipes, tape, soaker paper, tubes of water, markers, pens, post-it notes, etc. If you are in the habit of having a "waste container" for tips on your bench, toss it. Use only when needed and empty it promptly.

Good luck