How do I dilute DMSO 0.1% to 0.05% in cell culture media?

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.)

Do you have any idea how to get rid of these bubbles? The cells are not dying, but they look unhealthy with these stress bubbles on top of the colonies.

(using ITS regions)

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?

I need collaborators from South Africa and Delhi. I am working on "Genome sequencing of beneficial microbes for enhanced production of industrial and pharmaceutical solvents". Collaboration from Research laboratories for genome sequencing from these two countries are needed also. Thanks.

Dear all,

I am currently working with Caco-2 cells and need to culture them to achieve high cell numbers. However, due to my protocol, I cannot use trypsin for detachment. My main challenge is that when I collect the cells using a cell scraper, they tend to form visible, gel-like clusters. Despite extensive pipetting and gentle vortexing, these clusters persist, making it necessary to reseed them in this state.

While the cells still grow, it would be preferable if they were more evenly dissociated. Is there a way to overcome this issue and achieve better cell dispersion?

P.S. I have found a solution for counting the cells: I take 0.5–1 mL of the collected cell suspension and incubate it with trypsin in a tube. Once the gel-like clusters disappear, I proceed with counting. Since cell viability is not a concern for me, I incubate them with 1X trypsin for a longer duration than standard protocols suggest.

We have an antibody that we received as a gift from another lab (not commercially available) and the liquid has evaporated, leaving a sticky substance at the bottom of the tube. Is it possible to add liquid to use the antibody again? If so, what kind?

Hi everyone. For the purpose of my masters thesis, I need to develop a methodology to thaw a plant-based yoghurt-alternate sample which is preserved at -80°C to carryout microbiological analyses. Can anybody suggest a methodology or a concept to freeze and thaw in which least disturbance is caused to the sample? Note- once thawed it need not be refrozen. Thank you in advance.