Yesterday I incubated some of my competent H. influenzae cells with DNA carrying a novobiocin-resistance gene. One tube got the normal prep of chromosomal NovR DNA. Two others got DNA that had been cut with one of two restriction enzymes (either EcoRI, average fragment size 6 kb or XhoI, average fragment size 12 kb) and then had biotin incorporated at the ends of the fragments, and three others got streptavidin-coated polystyrene beads with the biotinylated DNA bound to them.
I had calculated that the first batch of DNA+beads had about 250 ng of DNA per ml, so I used the same volumes of beads for the others, and diluted the non-bead DNAs to 250 ng/ml before using the same volumes. I incubated the DNA plus cells for 15 minutes and then plated the cells on Nov agar, and (diluted) on plain agar. Today I counted the colonies and calculated the transformation frequencies.
Point 1. Cutting the DNA with XhoI reduces its transforming ability by 4-fold, but cutting with EcoRI reduces it by about 100-fold. So I checked where these two enzymes cut relative to the NovR (gyrB) gene. EcoRI cuts inside the gene, but XhoI only cuts at sites 1 kb and 3 kb on either side of it.
Point 2. DNA attached to beads transforms! This wasn't a sure thing, for lots of reasons.
Point 3. DNA attached to beads transforms 10-30-fold worse than the same amount of free DNA. This is not surprising because most of the DNA on the beads will be inaccessible because it's tangled up with or behind other DNA fragments.
Now I'll celebrate by spreading some poly-L-lysine on some cover slips, using the new method I was taught yesterday, so tomorrow I can test whether cells bind to these and whether they stay alive after binding.
- 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 Corrections5 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.
1 comment:
Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="http://www.fieldofscience.com/">FoS</a> = FoS
Subscribe to:
Post Comments (Atom)
Rosie:
ReplyDeleteIf EcoR1 cuts inside the gene, then where do the Nov resistant transformants come from? Are they taking up multiple pieces of DNA and making a functional gene through repair? Is the EcoR1 digestion incomplete?