Hello everyone,
I am seeking advice on a persistent issue with my microbial 16S PCR amplifications with primers 515F-Y and 926R. Despite extensive troubleshooting, my reactions consistently yield two distinct bands at approximately 500 bp and 700 bp, whereas protocols describe a single fragment range ~410 bp.
I have already attempted to resolve this by optimizing the annealing temperature. As shown in the attached image, a gradient PCR from 50°C to 55°C did not eliminate the double band. The problem persists under the following reaction conditions: 25 μL reactions, with Standard Taq (tested for cost) and Phusion High-Fidelity (same result), 0.25 mM dNTPs, 0.3 μM each primer (with R1 and R2 tails for a nested PCR approach), 2 mM Mg2+, and 5 ng DNA template; thermocycling in a ABI Veriti 96 Well equipment with initial denaturation at 96 °C for 3 min, 30 cycles at 96 °C for 40 s, 50-55 °C for 40 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 5 min.
This issue is halting my PhD student project, as I am reluctant to proceed with sequencing what appears to be a non-specific product, especially with limited funding.
I am aware of a similar discussions on ResearchGate, in particular this one here (https://www.researchgate.net/post/Double_band_on_16S_PCR_using_v3-v4_region_how_normal_is_this), which ended without a definitive solution.
Has anyone encountered and successfully resolved this specific problem?
Any insights into potential causes, such as primer dimers, secondary structure, or contaminating DNA, would be greatly appreciated.
Thank you in advance for your time and expertise.
Try following steps might be it will affect your results
1. Try to reduce primer concentration and also check primer contaminations.
2. Verify reagent purity.
3. Use negative control.
Hi Thiago,
when I see it correctly you analyse environmental DNA. Here you can have prokaryotes as well as eukaryotes - please be aware that primer pair 515F-Y and 926R amplifies 16s rRNA as well as 18s rRNA. THe average amplicon size for 18s rRNA is described with around 600 bp - this means around 200 bp larger than the amplicon for the 16sRNA amplicon - this would fit to the size difference you see on your gel - and this might be not so well known for this reason people miss this fact.
If you have prokaryotic and eukaryotic DNA in your environmental DNA it should be expected that you get 2 PCR products with only one PCR (please check out these publications: Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples - Parada - 2016 - Environmental Microbiology - Wiley Online Library and Comprehensive single-PCR 16S and 18S rRNA community analysis validated with mock communities and denoising algorithms).
In addition I think on your gel the exact amplicon sizes are hard to estimate - I would advice to let the gel run longer, use a higher percentage gel or try out an polyacrylamide gel for better resolution to see how large your PCR products really are. You could also try to subclone your PCR products in a suitable vector and send clones for seqences to see if the lower PCR band is due to 16s rRNA and the larger one due to 18s rRNA.
I am optimistic that this might be the answer to your question and that you do everything right methodically.
Good luck
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