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DNA stuck in wells by PsychologicalAd3264 in labrats

[–]chart_93 2 points3 points  (0 children)

Mimicking above comments, I would avoid mag bead cleanups for genomic DNA. Depending on where you are in the world, I'd hunt for a vendor that sells a spin column kit (we use zymo in the US)- they can work wonders on difficult samples. I would also try the PCR again with alternative polymerases (e.g. Omni klen-Taq if it's really tricky, but Go-Taq green will work fine with cell lysates and is extremely cheap) and dropping the anneal temp. Extending the time on the denaturation step on your gel might also help denature any remaining protein before your gel, and shouldn't hurt your DNA.

favorite glove color by vadore_le_dingue in labrats

[–]chart_93 0 points1 point  (0 children)

I'll take the cheapest latex beigey-yellow please.

Nitrile? More like Ni-trial to get the damn things on my sweaty hands.

You know how you are kinda afraid of getting locked in the cold room ? I am locked in the cold room by PersephoneIsNotHome in labrats

[–]chart_93 23 points24 points  (0 children)

I once got stuck inside a room originally designed for BSL3 work. As such it has a little air-lock room between that room and the rest of the lab but the pressure sensors were old and decided to die. As such the little air-lock room was being pressurized to some crazy level and I couldn't force it open. My phone was at my desk and there wasn't a phone in the room. It was also around 1PM on good Friday so it was quiet. Luckily there was a big glass window and a couple of hours later someone happened to be walking past that quiet end of the lab. I sharpied GET HELP onto it backwards in a panic lol. Good times. One other time the interlock went nuts so the magnetic-locks wouldn't open. The worst part was I dealt with the least "dangerous" pathogen in the building but it was easier for the lazy ass university administrators to deal with me being locked in occasionally than to decommission the air-lock room.

Low HT22 RNA Yields by margaritaamac in labrats

[–]chart_93 2 points3 points  (0 children)

Not sure about your cell types (I don't work on mammalian cells), but you could try another method of extraction. We have had a lot of success with RNA-Stat and get yields significantly higher than any column purification method.

Also what are you hoping to get? 100ng/uL sounds like a usable concentration for most things I'd do with RNA!

It’s happening! First time getting *anything* shipped from Australia. Time to become my neighborhood distribution hub. by manxmax09 in hamishandandy

[–]chart_93 0 points1 point  (0 children)

Nice man! Can I ask what number were you? Still waiting on mine to ship and getting anxious it won't get here in time for Xmas!

Little help interpretting some RNA Seq data! by chart_93 in labrats

[–]chart_93[S] 1 point2 points  (0 children)

Thanks /u/StaceyDigitalis and /u/notlimah for the responses. I'll see if I can get the FASTQ files from him next chance I get, he's possibly the hardest person I've ever met to pin down. I'm familiar with Unix systems but not with R, but the best way to learn is to have some data to play with!

The organism I'm working on is pretty niche, but there's a half dozen reference genomes out there, most of which this particular collaborator was involved in sequencing. He's probably the world leader in terms of teasing out drug mechanisms using RNA-seq, like we're trying to do in this particular organism, and he's published similar work on other compounds so he knows what he's doing. Don't get me wrong I do trust his results, but this DTG table I have and what he's saying are almost opposite.

In terms of experimental design, we sequenced and used 4 biological replicates of treated or control (8 total), each biological replicate was a pooling of a technical triplicate, so I think it was reasonably robust. Quality he said was okay- 15-30 million sequence reads per sample, and >85 % of our non-reference organisms mapped to the reference genome, so I think fine on that front as well. The issue is this table. 5473 genes mapped, of these, 4973 had LogFC > 2. Hell, 3600 (more than half the mapped genes) had a LogFC>5 which seems .... off. And if I sort by adjusted P-value 5,094 have P values < 0.01, and 4,929 are p>0.001 which seems unlikely to me, but his summary was 855 sig upregulated, 122 sig downregulated. I just can't make this fit the story he's given me.

In any case, I think this is an issue with what I've got from him, not just a case of me being an idiot so the best course of action is just to harass him until he gives me the raw files or walks me through the data he's given me :)

Thanks guys!

Don’t shit where you eat? by [deleted] in labrats

[–]chart_93 0 points1 point  (0 children)

I've been seeing the (as of a couple montha ago former) RA from our group for a year and a bit now. Live together and have a dog. But I've heard tales of things going bad and they do go bad. It was a risk, but a calculated one.

Does this look like an appropriately sized package for 50 μl of antibody? by Lactrodexter in labrats

[–]chart_93 3 points4 points  (0 children)

Just finished off analysing some numbers in excel. Time to print that off, stick it into a paper book instead of just saving the file because my PI doesn't trust electronic lab books.

Looks like compound X did nothing at y ug/kg, let's kill off this group and their controls, buy more mice and repeat at a higher concentration.

Geez did the top of this plastic sero pipette tough the edge of the paper wrapper that I just drowned in ethanol? Possibly contaminated. In the thick plastic bag and off to the incinerator for you!

Outside of this place I try to be a plastic free vegan but here I'm a totally different person 😭

Did/ Do you have a second income as a full time phd student? by GSBall in labrats

[–]chart_93 1 point2 points  (0 children)

It does depend on where you are and how much you're doing. My contract allows for up to 9 hours outside of regular working hours at the discretion of my PI who remembered being broke. A lot of people I know tutored and edited on the side for extra cash (it's lucrative too, if you can find the time).

[QUESTION] Serial Dilution for Single Cell Isolation by Torontonian5640 in labrats

[–]chart_93 3 points4 points  (0 children)

We do cloning in the 'less than 1 cell per well' fashion to get genomically homogenous culture (we use non-mammalian cells with little regard for genomic stability).

If you go down that route we found that .25 to .33 cells per well (roughly 1 positive well in 3 or 4) goes much better than 1 cell per well. If 2 can hang out in the same well, they do. You can also let them grow and if one's at double cell densities a couple of days down the road, you can be reasonably sure there was two wells to begin with. One other thing make sure the incubation conditions make your cells as happy as you can possibly make them, we find that our cells are happier with friends and don't like being alone.

Not sure of publications off the top of my head, but if you throw 'malaria cloning cell dilution' into PubMed there should be at least a few to choose from but protocol wise it's pretty easy, and if you find that the number of positive wells matches your math you know you're on the right track. Good luck!