Okay, I know the obvious answer is primer dimers, but I looked at the melt curves, and I am seeing the expected Tm.
But I have been getting amplification in my NTC controls in my qPCR. The Cts are high (30-35), but it messes with my run, because the genes I am trying to quantify are present in very low amounts. The strange thing is that I am only seeing this in 3 out of 4 of my primer sets.
HKG#1 is totally fine, no amplification at all. Just as expected.
HKG#2 shows extremely high contamination (Ct of upper 20s)
GOI#1 contamination around 35 Ct
GOI#2 around 37 Ct
I diluted the GOI primers 1:10 and 1:100, and tried again, and that seemed to get rid of it, but I'm worried that primers that dilute might not pick up a signal in my RNA samples.
I use clean gloves, filter tips, diluted new primers, used new water, used new SYBR green mix. I wouldn't be as confused if it was in all of my primer pairs.
My HKGs are all published primers, and my GOI primers were made using Primer3. I have ran gels looking at my GOI primers, and seen only one band at the expected size.
What is going on here?
I forgot to add that I have 3 sets of primers for each of my GOIs, and each set is showing this contamination. ( I have read enough troubleshooting to know that the first recommendation is design new primers.) The two genes of interest are homologs, so designing primers to differentially amplify them is trickier than normal.
First, I would not dilute your primers that much, you will not get a good signal if you do. Make sure you are using a positive control in every run so you can make sure your assay is detecting properly.
Here are a few questions that might help narrow down the culprit:
1) What water source are you using? (Bought, in-house purified etc)
2) Did you re-suspend all of your primer stocks with the same water at the start? (The contamination might have arisen at this stage, and if it did, getting new aliquots from the same stock will not rule it out)
3) Are the genes you are working on highly conserved?
4) Are you using latex gloves with powder? (The powder is most often derived from wheat, and I'm guessing from your profile that you are working on plant assays)
5) Have you tried a new batch of mastermix? (Looking for contamination of the mastermix tube)
6) Have you tried a different brand of mastermix? (Looking for background DNA within the mastermix itself (not unheard of))
Thanks for the tips.
1. Our water is bought, so I'd hope it's not the culprit
2. All primer stocks have been diluted with the same water. Even the primer set that is not showing contamination.
3. The genes I am working with are somewhat conserved (80-90% homologous) so designing new primers is a real chore
4. I was using latex gloves, but I'm not sure if they are powdered or not. But I will try with nitrile gloves next time
5. I have tried a new tube of master mix
6. Will look into a different mix... Maybe a different lab would let me try a few reactions, before buying a whole tube.
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