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The DAN lab
United States
Registrace 1. 03. 2021
The DAN lab is a research group at the University of Wisconsin-Madison led by Dr. Daniel Amador-Noguez, focusing on questions of microbial metabolism. We are here to share our science and lab techniques! Check out our website or contact us for more information.
Phage isolation and concentration by cesium chloride (CsCl) gradient
CsCl gradients are a long-used way of separating complex mixtures by density. Here, we demonstrate a protocol using a density gradient to isolate phages at high concentrations. In addition to concentrating large quantities of a single phage species, as we show here, phages with sufficiently different densities can be separated from one another using this technique as well.
This protocol relies on ultracentrifugation. Please ensure proper training and exercise caution when using an ultracentrifuge.
Music by bensound.
This protocol relies on ultracentrifugation. Please ensure proper training and exercise caution when using an ultracentrifuge.
Music by bensound.
zhlédnutí: 2 887
Video
Phage Hunting
zhlédnutí 209Před 2 lety
We're out and about, on the hunt for novel phages! We were hoping to find phages that infect the bacteria Zymomonas mobilis, since to date none have been discovered. We think Z. mobilis might live on apples, so come along to some local Wisconsin orchards to see how we did our search! NOTE: Z. mobilis is a completely harmless bacterium! It does not cause disease. It ferments sugars into alcohol ...
Biofilm growth in 12-well plates (B. subtilis)
zhlédnutí 8KPřed 2 lety
Protocol for growing Bacillus subtilis biofilms in 12-well tissue cultures plates. We use stainless steel mesh inserts to make it quick and easy to remove the complete pellicle. We use this technique for mutant characterization, metabolomic, and lipidomic investigation of biofilm formation. Music by bensound.com We study microbial metabolomics at UW-Madison - check out our website! danlab.bact....
How to: Change a gas tank
zhlédnutí 952Před 2 lety
This video covers tank safety for the gas tanks used around the lab, and then shows you step-by-step how to change out an empty tank for a full one. Tank safety: 0:10 - 4:25 Swapping tanks: 4:25 - 7:15 Safety reminders: 7:15 - end We cannot emphasize safety enough! Grab a labmate for assistance if you feel hesitant or haven't moved a tank by yourself before. Music by: bensound.com
How to: Change the HPLC column (Q Exactive Orbitrap)
zhlédnutí 1,6KPřed 2 lety
Demonstration of changing the HPLC column for the HPLC in the newer DAN lab LC-MS system (Gromit). For changing solvents, go to czcams.com/video/T0jmzJdPDok/video.html Music by bensound.com
How to: Change HPLC solvents (Q Exactive Orbitrap)
zhlédnutí 1,2KPřed 2 lety
Our DAN lab manager/HPLC extraordinaire demonstrates the practice of swapping out solvents for our newer HPLC-MS system. This video is intended for use as a refresher for using our instrumentation, not a substitute for in-person training Music: bensound.com
How to: Making solutions anaerobic with a gas manifold
zhlédnutí 2KPřed 3 lety
Making anaerobic solutions is an essential part of our work in the DAN lab! Many of our bacteria prefer to grow without oxygen, and some cannot grow at all unless we remove all the oxygen from their environment! Here we walk through the gas manifold system that we use to replace the oxygen in our tubes and medias with nitrogen gas. Notes: 1) BE VERY CAREFUL when inserting stoppers into bottles!...
How to: High precision scale
zhlédnutí 906Před 3 lety
Hey there, DAN lab! Here's a tutorial on using the high-precision scale in the lab. This scale is great for being super precise when weighing tiny quantities.
How to: Cycle gas in the anaerobic chamber
zhlédnutí 1,1KPřed 3 lety
How to cycle gas in the chamber: step-by-step When H2 is low, an alarm goes off in the chamber. To fix it, you need to release some of the gas in the chamber and replace it with the mixed gas (5% CO2, 5% H2, balance N2). To do this: 1) Cycle the airlock to make it anaerobic 2) Open the inner door of the airlock (open airlock to chamber) 3) Turn off the airlock with the switch on the front 4) Re...
Getting a representative "snapshot" of metabolism
zhlédnutí 150Před 3 lety
An important part of studying metabolism is metabolite collection, and one concern we talk a lot about is knowing that the metabolites we are collecting are giving us an accurate picture of what is happening at the cellular level. Our lab at UW-Madison makes sure that we extract cellular metabolites without changing the environment that our cells are in, let's talk about it! TRANSCRIPT: Hi! I’m...
How to: Use a sample concentrator to dry solvent samples
zhlédnutí 1,2KPřed 3 lety
After we extract bacterial metabolites in organic solvents, we evaporate off the solvent and resuspend the metabolites in water for HPLC-mass spectrometry analysis. Here, we demonstrate how to evaporate off the solvent using a sample concentrator and nitrogen gas. Transcript: This is the 36-slot sample concentrator. The nitrogen tank located next to the hood is attached to the sample concentrat...
Metabolite Extraction from Bacterial Culture
zhlédnutí 5KPřed 3 lety
Demonstration of our protocol for collecting metabolites from bacterial cultures. For more info on accurate sample collection, try czcams.com/video/Uig9L7q65CA/video.html Transcript: Welcome to the DAN lab! Today we are demonstrating our protocol for extracting intracellular metabolites from bacterial cultures. This protocol requires some preparation before you dive in. To make the extraction s...
Wow, centrifugation is cool. Thanks ❤
Awesome video. Helped me concentrate my phages
This was a great and instructive video! I was having problems with phage purification, and you guys helped a lot!
Hello, I also want to assemble such a deoxygenation device in my laboratory. Do you have any detailed reference materials to share?😃
Maybe lose that mentally draining and inane tune playing in the background.
Please what type of filter paper was used for the extraction? My current research is on extraction of bacteria metabolites
I was told Water is not good on GCMS column if the ratio is 2:2:1 how is the water removed from the extraction solvent
Msgg means??? And explain about your results. How it appears??
I would have to keep watching this until I get it down packed. Is this the only way to grow?
5 decimals places my mind blown that's nuts!
Brilliant, thanks. We are testing the hypothesis of EET to the metal mesh and seems to work well. Can I contact you via email?
How can we go about analysing is extract is it better to use lcms or GC ms if my goal is searching for a antibiotics produced by the bacterial colony
Why there is bead in bacterial culture. Should we need to do bead mill method for cell wall disruption before this metabolite extraction?
No bead milling necessary! In fact, adding any steps in between growth and extraction will negatively impact your results - you want to extract cells in exactly (or as near as possible) the condition they were growing in. Changing the cells' environment will alter metabolites, even on the scale of seconds or minutes. The "bead" you see is just a stir bar, it's there because the bacteria I was growing tend to flocculate.
What type of mesh (size, dimensions, etc.) did you use for biofilm growth? Did you make the mesh yourself, or is there a commercial source? Thank you!
Great question! We buy stainless steel mesh as a sheet and then cut out the inserts ourselves using wire cutters. We cut it to fit whatever plate we're using, so we usually trace a well using marker, or make a small stencil out of cardboard to make it a little more accurate. It's a bit slow and tough on the hands, but once you have inserts cut out you can autoclave and reuse them over and over!
congratulation! 😀 I used to work with a similar device at University of Warwick_ UK