OK, after some delay due to putting the microtiter plate wrong-way-around into the plate-scanning machine, I have the results of the DNA assays of my bead+DNA preps before and after a final wash.
I think the diagram is pretty self-explanatory. Most important result: Almost all of of the DNA is firmly attached to the beads. Even for the magnetic Dynabeads (columns on the right), the volume of DNA-containing supernatant remaining with the washed beads contributes no more than 1% of the total DNA in the -after-wash prep. The three preps on the right have fewer but larger fragments than the three preps on the left.
Surprisingly, the Picogreen assay detected consistently more DNA on the beads after washing than before washing. This is probably an artefact of something. The beads were all originally in TE (10 mM Tris, 1 mM EDTA), and were washed in TE overnight and resuspended in TE. Might the overnight rolling at 37 °C have better dispersed the DNA so the Picogreen could access it easier? This seems unlikely to be the explanation as, because of my putting-the plate-in-backwards error, all the samples were mixed with Picogreen and then sat for 24 hr before being read.
Because the tweezers work doesn't need millions of beads, these preps should be enough to go on with. This characterization also gives me confidence that my biotin-tagging and bead-attaching procedures work reliably.
Next steps: Repeat the transformation and cell-attachment assays. But first, clarify the bead-clumping situation.
- 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.4 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.
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