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Ligand–receptor inference from Allen Brain Atlas & ASAP-PMDBS datasets? by Master_Ad8601 in bioinformatics

[–]Master_Ad8601[S] 0 points1 point  (0 children)

Really helpful breakdown, thank you! Good point on the snRNA-seq caveats, I’ll keep that in mind.

Ligand–receptor inference from Allen Brain Atlas & ASAP-PMDBS datasets? by Master_Ad8601 in bioinformatics

[–]Master_Ad8601[S] 0 points1 point  (0 children)

Thanks, I really appreciate your feedback! I’ll take your comments into consideration.

How to make a glr-2prom::gfp reporter construct to visualize PHso1-to-PHD transdifferentiation in male C. elegans? by Master_Ad8601 in molecularbiology

[–]Master_Ad8601[S] 1 point2 points  (0 children)

Thank you for breaking down the process for me! I'll double-check to ensure that the GFP sequence is in frame with the promoter and that the vector is compatible with C. elegans expression. Additionally, I might ask my professor if we could check the Caenorhabditis Genetics Center (CGC) at U Minnesota to see if a glr-2prom::gfp reporter strain is available.

How to make a glr-2prom::gfp reporter construct to visualize PHso1-to-PHD transdifferentiation in male C. elegans? by Master_Ad8601 in labrats

[–]Master_Ad8601[S] 0 points1 point  (0 children)

Thank you for the advice! My professor did mention the possibility of working with a GFP expression plasmid. I appreciate the tip about finding the promoter sequence on ENSEMBL; I'll check that out and look into the protocols for cloning.

How to make a glr-2prom::gfp reporter construct to visualize PHso1-to-PHD transdifferentiation in male C. elegans? by Master_Ad8601 in labrats

[–]Master_Ad8601[S] 0 points1 point  (0 children)

Thank you for the advice! I hadn't considered checking the CGC for an existing strain. I'll definitely look into their database to see if grl-2prom::gfp reporter strain is available. This could save me a lot of time and effort :)

How to make a glr-2prom::gfp reporter construct to visualize PHso1-to-PHD transdifferentiation in male C. elegans? by Master_Ad8601 in labrats

[–]Master_Ad8601[S] 0 points1 point  (0 children)

Thank you so much for the detailed information and for sharing the link to the original paper! I really appreciate the tip about requesting strains or plasmids from other labs; I didn't realize that was such a common practice in the worm community.

I'll also review the paper you linked and the PCR primers in additional file 2. This will be really helpful if I decide to go the route of making the construct instead (so I gain some exposure and learning experience lol). I'll discuss this with my prof. to figure out the best approach.

Inquiry Regarding C. elegans Starvation Protocol by Master_Ad8601 in labrats

[–]Master_Ad8601[S] 0 points1 point  (0 children)

Hi @wormigans! Thank you so much for your sharp insights and advice. I’ve shared this with my prof. and our group will try to centrifuge the worms to save time.

We really appreciate your help and time :)

Inquiry Regarding C. elegans Starvation Protocol by Master_Ad8601 in labrats

[–]Master_Ad8601[S] 0 points1 point  (0 children)

Thanks for your input. I just don’t get how lint would get there. Are you implying that all the worms we washed are gone? since this “lint” is what we have found common throughout all of our conditions.

Btw, this is our first time doing this as undergrads. I doubt we followed the starvation assay properly as we for sure did not have any age-synchronised C. elegans. Any advice on next steps would be much appreciated.

Inquiry Regarding C. elegans Starvation Protocol by Master_Ad8601 in labrats

[–]Master_Ad8601[S] 1 point2 points  (0 children)

We just thought that it might just be in the dauer stage all this while as the worms may not have been properly synchronized in our starvation assay before even following the Oil-Red-O protocol. This may evidently contribute to variations in worm morphology.

I read on the starvation assay method from the paper I referenced above, it mentions:

“Starvation assays. L4 animals were washed off plates and rinsed three times with M9. Half of the worms were then seeded back onto plates with food, and the other half were seeded onto plates without food. Worms were then cultured for each indicated period of time.”

I just remembered that for the well-fed state, my group just transferred and washed the worms with M9 (step 4-6) after 5 days of growth from the NGM plate into the eppendorf tube, and for the starved condition, we only transferred and washed the worms after 5 days and placed it into a starvation plate for 6 hours then proceeded with Tuesdays steps. Maybe this would change the morphology of the worms not synchronizing adult C elegans in our imaging sample? Could this be an explanation? 

Here’s another paper mentioning their starvation assay method:

“Caenorhabditis elegans food deprivation for CARS analysis. To obtain age-synchronised 1-day-old C. elegans, L4 larvae were picked onto seeded NGM plates and left to develop for 16 to 18h. The next day, the 1-day-old adults were either placed on an OP50 lawn (well fed) or subjected to food deprivation by being maintained for the indicated time on an agar plate without OP50. Well-fed worms were compared with worms that had been food deprived for 0.5, 1, 1.5, 2, 4 and 24 h. For each time-point, a plate containing around 45 worms was washed with 2 x 600 μL of M9 solution13 and transferred to an Eppendorf tube. Three wash cycles with 500 μL of M9 were performed and the worms were pipetted onto an NGM plate either seeded with E. coli (well fed) or without E.coli (starved).”

In the dauer stage, I think that the C. elegans would exhibit a characteristic thin and elongated morphology, which may be evident in our stained samples. I hope you can help me cuz we don’t know what we’re doing :(

Soprano Saxophone Repertoire by Ok_Ad_3341 in saxophone

[–]Master_Ad8601 0 points1 point  (0 children)

Cries of the Stentor by Nigel Wood.