What is the minimum percentage of identity required by BLAST to placement into genus and species?

I have a problem that no bacteria bands appear, although I previously replaced the master mix, nuclease free water, and even used new primers, but the result is the same as in the pictures. Note that I took a PCR device to another lab to make sure, and it was fine and gave results. When I used it in my lab, it did not give results, as in the pictures. By the way, I tested the DNA genome only by electrophoresis, and the results were good, but after amplification by PCR, the results were as in the pictures every time, and I even used RNase to no avail.

Hi all,

We are currently working on extracting microbial DNA from clinical blood samples (from BacT/ALERT bottles) for downstream NGS applications. We’re using a commercial kit for microbial DNA extraction but are encountering issues with obtaining pure DNA. Despite performing additional purification steps, we are unable to achieve the recommended 260/230 ratio within the desired range.

We’ve tried multiple kits, including the Macherey-Nagel NucleoSpin kit, the Invitek DNA extraction kit, and the MP Biomedicals kit, but we still can't obtain the desired purity.

Could anyone suggest ways to obtain purer microbial DNA or provide insights on whether the blood samples from BacT/ALERT bottles might be causing any issues? Any advice or suggestions would be greatly appreciated.

Thank you in advance, Sumana

Dear community,

At Dr. Halverson’s lab at Iowa State University, we have successfully developed a protocol for library preparation and a bioinformatic pipeline for bacterial 16S rRNA gene sequencing using the MinION platform. However, amplifying fungal DNA has proven much more challenging.

I have tested several primer sets (listed below) using different PCR kits, a range of DNA template concentrations, and modifications to PCR conditions (e.g., varying annealing temperatures, final extension times, and number of cycles). Unfortunately, I have not yet been able to obtain clear bands at the expected size on gels, using either fungal genomic DNA or environmental DNA samples (soil and rhizosphere).

I would be very grateful for any advice, suggestions, or protocols from anyone who has successfully amplified fungal amplicons for MinION sequencing—especially regarding primer choice, PCR conditions, or any troubleshooting tips.

Project Goal: We aim to amplify fungal communities from soil environmental DNA. We are open to using primer sets that target broader eukaryotic groups, as fungi tend to dominate eukaryotic communities in our samples. Eventually, we plan to use the newly published EUKARYOME database (DOI: 10.1093/database/baae043) for taxonomic classification.

Thank you very much for your support!

Greetings, everyone.

I'm currently optimising a protocol on eDNA extraction from freshwater samples for a species-specific assay. I want to test multiple workflows, including either filtration or centrifugation of water samples. (Both approaches have been previously shown to be reliable for the species of interest.) Here's the thing: for centrifugation, I plan to use at least 200 ml of water split into 50 ml falcon tubes. After centrifugation, the eDNA-containing pellets should be pooled into a single 1.5 ml microcentrifuge tube for DNA isolation. The thing is: how would you go about the pellet-pooling part? I've already tested it, and the best thing I've figured out is to carefully remove as much supernatant as possible and then gently aspirate the pellet into a large pipette tip. However, it feels somewhat unwieldy and I'm afraid this step may introduce unnecessary inconsistencies. If you have experience with this sort of pellet transfer and pooling, do you have any tips on how to get the best results possible?

(The already-published papers only mention that pellets were pooled, without additional specification. I plan to contact their authors to ask for details, but having some do's and don'ts from a larger number of people cannot hurt.)

I have a DMSO stock solution with a concentration of 0.1%. I want to dilute it again to a concentration of 0.05% which I will use in my cell culture media which I add with alfalfa plant extract with a concentration of 2.5%. how do I make the solution?

Easy, Reliable and cost effective laboratory tests available in India

I'm currently working on potential fungi species and I got 3 species from the sequence result. I already constructed a phylogenetic tree with these 3 species, as well as ingroup with the most similar sequence (thx to BLAST). However, my outgroup will always be placed in the middle of the tree and not on the rock bottom. My phylogenetic tree method is Bootstrap. When I use the original tree construction, the tree seems pretty fine, but the outgroup suddenly went to the middle of the tree with bootstrap. Is there anything wrong with my choice of outgroup? Note: all of the fungi are coming from Dothidiomycetes Family with order coming from Capnodiales and Pleosperales. My outgroup however, is Mytilinidion resinicola from the same class and from Order of Mytilinidiales. I checked it before and the outgroup distance is not too close and not too far from my sample.

Thank you so much, I'm still pursuing a bachelor's degree. Sorry for this long question 😭

Is anyone using the differential supercoiling anatomy of DNA for phylogenetics: Any ideas on Phylogenetic Signal in such Data?

Hi,

I have extracted DNA from some cells and eluted in buffer AE (Qiagen) which came with the kit. After this I have diluted the sample in nuclease free water to make up a specific concentration for downstream application. What should I blank the nanodrop with at this point? The sample has more water than buffer in it now so which is the most sensible?

Thanks in advance.