I'm still having problems with the E. coli cells that contain the DNA construct for the advanced tweezers experiments. The cells grew OK overnight on an LB+Amp50 agar plate but poorly in what I later realized was LB+Amp200 broth (too much ampicillin). I did a plasmid prep from these cells but got little or no plasmid (a faint smear only). Yesterday the postdoc inoculated another colony for me, into LB+Amp50, but this also stopped growing at a low density, and a plasmid prep gave no plasmid at all.
The E. coli cells are strain DH5alpha, which should be quite vigorous, and the plasmid is a derivative of pGEM and so should have a high copy number, even with a 12 kb insert. Perhaps the cells aren't what they're supposed to be. Maybe there's something wrong with my medium (and the postdoc's medium). Perhaps there was something wrong with my original LB+Amp plate. Perhaps the plasmid replicates poorly because it's so big. Perhaps the plasmid is present in the cells but lost during purification, because the miniprep kit doesn't give good recovery for DNAs bigger than ~10 kb.
Update: this morning i inoculated colonies from the LB+Amp plate into plain LB and LB+Amp50. The cells in plain LB seem to be growing faster, so maybe the plasmid is toxic. I'd better go back and reread the thesis chapter of the M.Sc. student who made it.
Field of Science
-
-
-
-
-
How dumb is too dumb? We still don't know!22 hours ago in The Phytophactor
-
-
The Even Earlier Discovery of Antibiotic Resistance1 day ago in Memoirs of a Defective Brain
-
Religion is halfway between a fact and an opinion - according to kids and adults2 days ago in Epiphenom
-
Bioengineers go retro to build a calculator from living cells3 days ago in The Allotrope
-
-
A New Non-mammaliaform Eucynodont from the Ischigualasto Formation of Argentina1 week ago in Chinleana
-
-
Chemistry, fluid dynamics and an awful radioactive mess1 week ago in The Curious Wavefunction
-
Exploding expertise2 weeks ago in The Culture of Chemistry
-
-
-
-
-
UPDATED: 10 things we need to find out about the #NCoV1 month ago in Rule of 6ix
-
-
-
-
-
The Lure of the Obscure? Guest Post by Frank Stahl11 months ago in Sex, Genes & Evolution
-
-
Finding a new translation factor, and verifying it with help from my experimental friends1 year ago in Protein Evolution and Other Musings
-
Free ImageJ Macro -- for citing images1 year ago in Skeptic Wonder
-
-
-
The Large Picture Blog Has Moved1 year ago in The Large Picture Blog
-
Lab Rat Moving House1 year ago in Life of a Lab Rat
-
Goodbye FoS, thanks for all the laughs1 year ago in Disease Prone
-
Branson getting into microbial diversity in the deep sea2 years ago in The Greenhouse
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.
4 comments:
Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="http://www.fieldofscience.com/">FoS</a> = FoS
Subscribe to:
Post Comments (Atom)
As I recall, the plasmid itself was replicated poorly, but the cells grew as regular DH5alpha E. coli did. My yields from maxipreps were always lower than for short plasmids, but I can't remember any problems with cell growth.
ReplyDeleteThanks, I was hoping you might remember. The cells grew a bit slower in LB+Amp, but growth of both cultures flattened out early, at about OD=0.5. SO I suspect there's something wrong with the medium.
ReplyDeleteLet me know if there's anything I can look up from this end (lab notebooks, duplicate stocks, etc.)
ReplyDeleteLet me know if there's anything I can look up from this end (lab notebooks, duplicate stocks, etc.)
ReplyDelete