Hey Winnifred, it could be literally anything. Just give us a small summary of your experiments. You extracted microbial DNA from fecal samples, right? Which extraction method did you use? Did you get Amplicons from a Positive Control, ideally a positive DNA Extraction control, too? Did you measure the DNA concentration via Qubit, Nanodrop etc.? Did you run a gel or Bioanalyzer after your PCR? Which primer did you use, specifically? Did you make a Purification step with magnetic beads after the Extration or the PCR?

What is the latest step in the process that was successful?

Anything from sample handling to DNA extraction to PCR to gel electrophoresis could be an issue.

I'd suggest chatting with a colleague to help narrow down the list of possible issues & what to double-check.

Good luck!

The issue can come from many different factors which include:

Quality of DNA template: Impure DNA. Try using fresh prepared DNA or another method of isolation

Presence of inhibitors: Fecal samples have PCR inhibitors that reduce amplification. In the extraction step make sure the inhibitors are removed.

Primer issues: Primer concentration could be too low or unbalanced or not specific enough for the target sequence.

Thermal cycler issues: Might not be at the correct temperature or working properly.

You could identify the problem by using reagents in a control reaction with a template and primers that are known to work. You could also test the amount of control template and primers used in the reaction, or you can calibrate the thermal cycler temperature.

Thank you so much for your feedbacks.

I have used CTAB with beads and Quigen Soil kit for extraction of DNA. The quality was not so good (53ng/ul) DNA. I performed gel and there's indication of gDNA although for extraction with soil kit, DNA was smeared.

I did I6s PCR to amplify V3 and V4 using 27F and 1438R primers. I also used ITS primers, as well as Universal plant primers and plastids. I used Taq Polymerase so PCR conditions were set accordingly to manufacturers instructions for PCR for Alltaq. My there's amplification on the control, but none for gDNA.

I'm using CTAB with proteinase K only for lysis, Chloroform isoamyalcohol (24:1) and ethanol for Precipitation of DNA.

Hello, as for me, I conducted a research on Herbivore(elephant) diet from their fecal sample source.

I used Uniplant primers and noticed no amplicons on the first run. I then decided to just try out doing a second PCR on the amplicons using the same primer and check...(more like a nested PCR) and saw bands in the gel and a third one just to be sure, since the BP are almost close to primer dimers, I did a third PCR same primers on the 2nd amplicons, the bands were brighter...and when purified and sequenced, I found out the diet contents.

Thank you Tevin Onyango. Thank you for sharing your tips. Its great to know yours did work. Awesome!

Just one quick doubt here. Did you use the PCR product of UniPlant as the templete DNA to run second PCR?

Welcome, and hopefully yours works too🙏.

Yes I used the Uniplant product as template DNA to run the second PCR and for some had to do 3 runs maximum for the bands to be visible.

So for the ones with 3 runs, I used the 2nd Uniplant PCR products as the template DNA.

Attached is the gel run after the amplification.

Thanks Tevin. Great result!

Welcome.

Hi Tevin, did you use the same primers in the second PCR runs?

Yes yes

The primers were a single pair (Forward and Reverse) so I used the same primer.

Nested PCR as you know has primary set primers and secondary set primers...with the idea I just used the same primer for my case.