Now I have lots of biotinylated DNA, and a well-tested procedure for binding DNA fragments to streptavidin-coated styrene beads, I'm ready to test whether competent bacterial cells (B. subtilis or H. influenzae) will bind to the DNA on the beads.
How to do this isn't straightforward. One problem is that the beads are about the same size and density as the cells (B. subtilis cells a bit bigger, H. influenzae cells a bit smaller), so once mixed they can't be easily separated. That means I have no way to wash unbound beads away from cells, or unbound cells away from beads. Another problem is that B. subtilis cells are known to cut DNA fragments as part of the uptake process, and in principle this might terminate uptake. Though maybe not, as the cutting is part of the process that initiates uptake across the inner membrane. H. influenzae cells don't cut DNA.
I could just mix competent cells and DNA-coated beads, both at low densities, on a microscope slide and watch for them sticking to each other. Alternatively, we have some streptavidin-coated paramagnetic beads I could use - this would allow me to pull out the beads and see if cells had stuck to them. But these 50 nm (super-tiny) beads, too small to see individually, so I'd have to plate them to see if there were cells there. We might also have some micron-sized ones; I'll look around.
OK, I found our 'starter kit' of 1 and 2 micron paramagnetic beads. The only problem is, we were too cheap to pay the $150 for the starter version of the magic magnetic rack that holds microfuge tubes against magnets so the beads stick to the side and the liquid can be removed. So I tested various magnets from around the lab, and all of them pulled the rusty-brown beads to the side of the tube in a couple of minutes. Doing this in a way that holds the tube steady so I can remove the liquid...not yet.
I'm away for a few days, but when I get back I may send an email out asking if anyone in the building has a Dynabeads rack I could borrow for a little while.
- Home
- Angry by Choice
- Catalogue of Organisms
- Chinleana
- Doc Madhattan
- Games with Words
- Genomics, Medicine, and Pseudoscience
- History of Geology
- Moss Plants and More
- Pleiotropy
- Plektix
- RRResearch
- Skeptic Wonder
- The Culture of Chemistry
- The Curious Wavefunction
- The Phytophactor
- The View from a Microbiologist
- Variety of Life
Field of Science
-
-
-
Political pollsters are pretending they know what's happening. They don't.5 weeks ago in Genomics, Medicine, and Pseudoscience
-
-
Course Corrections6 months ago in Angry by Choice
-
-
The Site is Dead, Long Live the Site2 years ago in Catalogue of Organisms
-
The Site is Dead, Long Live the Site2 years ago in Variety of Life
-
Does mathematics carry human biases?4 years ago in PLEKTIX
-
-
-
-
A New Placodont from the Late Triassic of China5 years ago in Chinleana
-
Posted: July 22, 2018 at 03:03PM6 years ago in Field Notes
-
Bryophyte Herbarium Survey7 years ago in Moss Plants and More
-
Harnessing innate immunity to cure HIV8 years ago in Rule of 6ix
-
WE MOVED!8 years ago in Games with Words
-
-
-
-
post doc job opportunity on ribosome biochemistry!9 years ago in Protein Evolution and Other Musings
-
Growing the kidney: re-blogged from Science Bitez9 years ago in The View from a Microbiologist
-
Blogging Microbes- Communicating Microbiology to Netizens10 years ago in Memoirs of a Defective Brain
-
-
-
The Lure of the Obscure? Guest Post by Frank Stahl12 years ago in Sex, Genes & Evolution
-
-
Lab Rat Moving House13 years ago in Life of a Lab Rat
-
Goodbye FoS, thanks for all the laughs13 years ago in Disease Prone
-
-
Slideshow of NASA's Stardust-NExT Mission Comet Tempel 1 Flyby13 years ago in The Large Picture Blog
-
in The Biology Files
Not your typical science blog, but an 'open science' research blog. Watch me fumbling my way towards understanding how and why bacteria take up DNA, and getting distracted by other cool questions.
How best to test binding of competent cells to DNA on beads?
Subscribe to:
Post Comments (Atom)
No comments:
Post a Comment
Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="http://www.fieldofscience.com/">FoS</a> = FoS