SGR is a sample and PJFH1 is endogenous control.
*The mixture was the same for both samples, changing only the primers.
If this ladder is a 100bp ladder, your amplicon size are below 100bp (what amplicon size are you looking for), that implies that the sharp bands you are seeing are primer dimer (not amplicon/no product). If that is true, repeat the experiment with cautions on nucleic acid concentration, primer concentration (to avoid dimer formation), the annealing temperature, and retention time for PCR extension.
May want to check primer dimer on qpcr product from negative control (use no nucleic acid template+use two separate primer sets, separately) on a gel, or do a melting curve study as mentioned by Bru.
Even though NTC melt curve doesn't appear on the chart, it seems that nothing but primers dimers was amplified for SGR. Would you have some positive control, gDNA (if possible, according to the location primers were designed) or maybe the cloned target ? just to make sure that PCR set up is fine.
Here's the amplification curve to control using plasmid for the SGR in serial dilutions. We used the same primers in both experiments. For plasmid works fine, but when the cDNA is derived from the cell, no.
Ok, then, since GAPDH was succesfully amplified on cDNA, let's consider that their quality is ok. Concerning SGR, based on your serial dilution, the assay may properly work as well. It's most likely that your target's number is below the detection limit... maybe could you try with a higher concentration if possible. To my opinion, increasing the cycles number wouldn't solve this issue and would lead to differences in replicates.
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