Janet B. Matsen:Closed Lab Questions

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Lab Basics:


  • I still use 100% for everything.
  • 70% is still concentrated enough to be very flamable!

How does colony size vary with colony density?

    colony size varies with colony density
    colony size varies with colony density

Culture Media



Why do we grow E. coli on glycerol, not glucose?

  • Amanda Smith found:
    • "Glycerol play the part of a cheap carbon-energy source for E.coli.
      • Ecoli grown on glycerol excretes less acetate compared to e coli grown on glucose. (due to changes in the biochemical pathways and slower absorption rate of glycerol). Acetate is bad because it limits the growth of e coli. This is due primarily by methionine synthesis inhibition, which in turn disrupts protein synthesis and alters protein expression (A horror story if you are expressing proteins). Acetate also acidifies the media which also cause other changes in E coli physiology (such as reduced growth rate, altered K/Na ion and amino acid anions concentration. Which also has effects on protein synthesis)"

Colony PCR



What causes an overloaded colony PCR reaction to fail?

  • A: Thermostable polymerases require magnesium (link). It has been suggested that because DNA chealates magnesium, overloading a colony PCR may inhibit the polymerase. (11/2012)
    • I'd love to test that by titrating in extra Mg in a series of overloaded colony PCRs. It is also possible there is a crowding effect or unfavorable interactions with the cell lysate and the polymerase.


Should I pay extra to further purify large bp oligos?


My primers are yellow!







normal and "burnt" electroporation cuvettes
normal and "burnt" electroporation cuvettes
  • added 7/31/2012

How does the density of electrocompetent cells affect transformation efficiency?

electroporation: number of colonies versus competent cell density
electroporation: number of colonies versus competent cell density
  • It matters a LOT! The undiluted samples (dilution factor = 0) were harvested ad mid-exponential phase and resuspended in 1/500ths of the culture volume. Details of the experiment are here.

What is the ballpark efficiency of electroporation?

  • In the experiment described just above, I calculated that for 0.1 ng of plasmid, the maximum efficiency was 5*10-6. I haven't looked up literature values to see how this compares to published statistics. This statistic makes Gibson cloning seem even more amazing. Even if you get only a few colonies, there were likely many orders of magnitudes more viable plasmids in the transformation product than that!

Gene Expression & Plasmids









How stable is HEPES?

  • Not stable. Make it the day you use it.




Is it "media" or "medium"?

I have recently switched to using "medium" when describing a single type of solid or liquid growth substance.

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