Positive signals in negative controls (including distilled water and no-template controls containing only the primer master mix), coupled with the absence of amplification in some primer sets, may be primarily attributed to the following factors:

Recommendations: Implement additional negative controls throughout the workflow, freshly prepare and store primers and buffers in separate aliquots, optimize reaction conditions, and verify the specificity of each primer pair.The answer to this question is provided by MedChemExpress Technical Support.

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If you are seeing amplification of a product in your negative controls, then you have contamination.

I'd recommend you stop doing any bench work & talk with your advisor. You need to know exactly what size bands you would expect, what successful controls look like, etc. BEFORE you start pipetting anything. No point in wasting reagents when you don't know what data to expect.

Try working with just the controls for 1 set of primers until you can get those working. Then add in another set of primers.

You also need to know how you will analyze your data.

Good luck!

• Follow these:
• Inspect melt curves + gel on existing products.
• Prepare clean aliquots and set up the next run with mandatory controls (NTC, NPC, positive dilution series, housekeeping).
• Try simple optimizations: lower primer conc. to 200 nM (or 100 nM), raise Ta +3 °C, run gradient.
• If NTC same-Tm → decontaminate, use fresh aliquots, consider UNG/dUTP.
• If issues persist for a primer pair → redesign primers using the checklist and validate anew.