Hello everyone, I have the following problem: I'm observing a lot of smear after loading qPCR products on 1.3% gel (attached pUC19-M13_primers_test) eventhough the melt curves seem nice with only one peak (attached melt_curves_pUC19). In the reaction I've used 10-fold dilutions of pUC19 fragment (121 bp) using M13 primers (from 1E7 (St7) down to 1E4 (St3)). I've tried if different primer concentrations show any difference but to me it all looks the same. Standard curves of different primer concentrations showed between 89-92% efficiency. This was done on QuantStudio5 instrument, with 384 well block. So my questions are the following:
1. How come that only one peak can be observed on a melt curve while a smear of different products can be observed on a gel?
2. What is causing the smearing on a gel? If it would be the gel or any of the components of the electrophoresis probably also the ladder would be smeared or?
P.S. Primers were freshly prepared, the same goes for the plasmid dilutions, which were diluted from the commercial stock solution. Amount of DNA in the well with the highest DNA load was negligible (0.03 ng/uL) as calculted from the 1E7 copy number of pUC19 plasmid. Electrophoresis was run in 1xTAE buffer, the same buffer was used for preparing the gel.
Thank you very much in advance!
Have you tried amplifying your product using standard PCR (i.e. without SYBR Green) and running the product on a gel? That's always a good troubleshooting step.
Peter Chomczynski thank you very much for your answer. Could you explain a little bit more why this would be a good troubleshooting step? I'll try it, I would just like to understand the logic for the future :). Thanks in advance!
It looks like you have over-loaded your gel. The retained brightness in the wells means the salt concentration might be off as well.
I'm inclined to say you have one band and what you have in the gel is an artifact from overloading with too much sample & running the gel after the samples have been melted.
Melt-curves can also make DNA bands get "fuzzy" since the molecules don't always re-anneal properly.
The easiest way to see if you have one band is:
set up your qPCR reaction like usual, but do NOT include the melt-curve step.
Load part of each reaction in an agarose gel.
Use the rest for a melt-curve.
See what you get!
Dr. Katie A Burnette provided a more detailed explanation. Your melt curve looks good so I anticipate that your product is OK. You have a high quality instrument and SYBR Green qPCR is a very reliable method.
The reason I suggested a standard PCR & gel run is because it provides a secondary method of confirming your melt curve results, since that seems to be what you're trying to accomplish, without any potential interference from the qPCR + melt process. I would try loading 2 or 3 different dilutions of your template into the PCR reactions. This process will give you extra confidence that your method is working correctly.
Katie A Burnette and Peter Chomczynski thank you soo much for your help! I'll try it as soon as possible and post the results here.
Dear Katie A Burnette and Peter Chomczynski thank you for your help on the issue. As you can see in the attachement your advice worked and there is only a single band if I don't use a melt curve step. I didn't need to use the dilution to get this result. I didn't realize the melt curve was a reason for the smear since I was working with the same assays on Quiagen RotorGene in the past and this was never a problem.
I also raised a separate question regarding usage of BSA, DMSO and T4 gp32 in qPCR assays for soil microbial community, if you might have any ideas please let me know:
https://www.researchgate.net/post/How_to_decide_on_when_to_use_T4_gp32_BSA_and_DMSO_use_in_qPCR_for_quantifying_soil_microbial_community
Thank you again and good luck with your work!
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