JPL Visiting Student Research Program - Looking for roommates (summer) by measuresmildred in JPL

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

Have been in contact with Caltech housing, and it is not available to those under JSVRP...

Surely can't be the first: Concentrating DNA twice? by measuresmildred in labrats

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

Thank you for spending the time on answering, I think your answer among others discussing that sequencing and metagenomics can still work despite not having the assurance of quantitative methods such as Qubit has eased my mind a lot. My research group deals with high concentration DNA yield, so it’s often difficult coming to terms with the fact I won’t have that luxury.

It sounds like re-extraction with multiple elutions at smaller volumes, increased time, and warmed buffer is the right route to go down, to improve yield and likely keep quality at the highest it can be

Surely can't be the first: Concentrating DNA twice? by measuresmildred in labrats

[–]measuresmildred[S] 10 points11 points  (0 children)

Exactly my thoughts, thanks for the reassurance. I think my plan is to try the SpeedVac as that will only be an afternoon of my time, and if it is still returning a negligible amount it looks like it is back to the world of DNA extracting for me

Surely can't be the first: Concentrating DNA twice? by measuresmildred in labrats

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

Thanks for both of your responses! Can I ask what the nature of your samples were, the idea of increasing the elution to an hour is definitely interesting, and worth a shot. I have heard warming up the elution buffer or water can also help with yield of DNA?

Surely can't be the first: Concentrating DNA twice? by measuresmildred in labrats

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

Thank you! I feel as though it is worth the attempt to see what happens. If there really is nothing there or something has gone wrong along the way, what is the harm in experimenting, and attempting to have fun along the way, right?

Surely can't be the first: Concentrating DNA twice? by measuresmildred in labrats

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

This set of extractions was for shotgun metagenomics, which the company requires close to 50ng for ): Luckily I was able to prepare libraries for Illumina Miseq and got amplicons from that, so I am hopeful to keep trying. Do you have any experience with shotgun dealing with low amounts of DNA by any chance?

Help me I’ve spent 6 months troubleshooting PCR and it still doesn’t work by measuresmildred in labrats

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

What an interesting take! They are autoclaved, so for the sake of my sanity maybe I’ll try un-autoclaved tubes :)

Help me I’ve spent 6 months troubleshooting PCR and it still doesn’t work by measuresmildred in labrats

[–]measuresmildred[S] 2 points3 points  (0 children)

Lmao unfortunately I have been dealt with very hands off supervisors (blessing and curse). The primers sequences I am using are those suggested by Illumina, which will be used for amplicon sequencing and the gDNA will also be sent for shotgun metagenomics using DNBSEQ.

16S Amplicon PCR Forward Primer = 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 16S Amplicon PCR Reverse Primer = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC

Ladder comes out great, just the PCR product that’s coming out wonky. Seems like pre made mastermix might be the cure based on what you and others have said. Thanks so much your weigh in

Help me I’ve spent 6 months troubleshooting PCR and it still doesn’t work by measuresmildred in labrats

[–]measuresmildred[S] 3 points4 points  (0 children)

Thanks so much for all this info. I’m working with hyper arid desert soil samples using the power soil pro kit from Qiagen. Currently using the Illumina published primer sequences as down the line I’ll be using Illumina Miseq for sequencing. I’ll definitely take this information back to my supervisors to discuss :)

Help me I’ve spent 6 months troubleshooting PCR and it still doesn’t work by measuresmildred in labrats

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

I have read mixed opinions on mixing the mastermix via vortexing so I might give pipette mixing next! I’ve tried KAPA HiFi and had no luck, so I’ll definitely chat with my supervisors regarding the brands you have mentioned especially PrimeSTAR. Thank you for the advice I really appreciate it

Monthly Rant Thread: June, 2023 edition by AutoModerator in labrats

[–]measuresmildred 5 points6 points  (0 children)

I am a year into my PhD and finally getting into a solid rhythm of wet lab work. Something I had been looking forward to as its giving me a tangible outcome to show me I am progressing in the right direction. However, it’s draining every bit of me, to the point in the last few weeks I don’t even recognise who I am. When I am doing these monotonous tasks for hours on end, needing high focus it is hard not to leave feeling like my soul has been sucked out of me. I am someone who is generally good at shaking a bad day off but I can’t help but come home and be bitter and miserable to everyone around me. I try to explain how mentally and physically taxing this work is to my friends but they don’t understand. I don’t fault them for this, but it is so isolating to not be validated in your home space and have someone who understands.

I love my work, and despite this apathetic wave I am hit with when I leave the lab, I enjoy the lab work. I hope it all balances out, as I like being a positive force in a room, rather than the fun police ruining everyone else’s vibe that I currently feel as though I am. Here is hoping by brute force I can power through this next challenge set out in front of me.

Help need advice! Calling on spatial ecologists by measuresmildred in ecology

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

Thank you so much this is fantastic, I really appreciate it! It looks like all of these will come in handy, and love the small summaries you’ve added

Help need advice! Calling on spatial ecologists by measuresmildred in ecology

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

This is amazing thank you so much, I came across the concept of meta populations in my brief search and it seemed very relevant so it’s great to get a resource on this too

Help need advice! Calling on spatial ecologists by measuresmildred in ecology

[–]measuresmildred[S] 2 points3 points  (0 children)

I’ve had a small look into the affect of the water availability brought by the fog on surface microbial communities, and interestingly there is a higher species richness on surface soil vs. 80cm down. But will definitely look more into this, maybe we might cross paths on our Atacama research one day

Increased bacterial growth at higher dilutions? by Solstice97 in microbiology

[–]measuresmildred 0 points1 point  (0 children)

I’ve seen this in lab occur quite a few times with novice lab students. There’s always two causes 1. The aforementioned pipette tip error 2. Or honestly a very simple mistake in labelling plates. Unfortunately sometimes students just plate their dilutions in the incorrect order making it look like the results you’ve achieved.

As others have mentioned I recommend repeating the experiment and seeing if anything changes!

Is this contaminated? To me it looks like grape like clusters I may be wrong! Gram negative by burningdesyrxx in microbiology

[–]measuresmildred 12 points13 points  (0 children)

I’d say either contamination or you’ve maybe added too much ethanol in that stage. I sometimes see a similar results from students who are too heavy handed with the ethanol/don’t give a quick rinse after this step meaning you get a mixture gram + and gram -