I am doing a gradient PCR to achieve standardization of my primer set, however the results I have obtained from it are inconclusive.
What do you recommend me to do?
The image shows tm tests from 50 to 60°C located upward from the first well
What size product were you expecting? Unless it was smaller than the smallest band on your ladder, I don't think anything amplified.
I'd suggest getting a reliable positive control DNA and try again.
What are the dna samples. Have you checked od260/280 ratio for dna purity and how much (ng) dna are you adding to the pcr reaction. At least one primer is working and probably both in the early samples and there is no obvious primer dimer on these samples so it looks like the assay is nearly working. What are your pcr cycle parameters and what voltage are you running the gel at?
what are the Tm s of your primers.
Are your primers new or have they been stored from previous researchers for a long time. Do you have a primer set for a housekeeping gene that you can run to check that your cDNA is of good quality?
There are several reasons might be behind this issue and I can suggest:
-Check the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions.
-If the negative-control PCR shows a PCR product or a smear, exchange all reagents. Use disposable pipet tips containing hydrophobic filters to minimize cross-contamination.
-Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.
-Reduce the number of cycles in steps of 3 cycles.
-Perform PCR with different final concentrations of Mg2+
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