User:Daniel Goodman/Notebook/Cluzel/2010/02/24

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Cluzel Lab Notebook
Daniel Goodman
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Redo glass experiment from 2/22

Prep

  • 3% w/v agarose
    • 5 ml LB, 150 mg agarose
    • heat in 80 deg. bath for 15 min
    • 108 ul per mold
  • 6 agarose block

Agarose Mold

  1. Mold agarose into blocks
    1. Not using silicon molds, only testing bacterial dispersion on agarose/glass slide.
    2. waiting 20 min for agarose to dry in PDMS molds, at room temp; in future, will try different drying temps/dessication?
    3. cover agarose in mold with cover slip

Cells

  1. Grew up wild-type MG1655 E. coli (w/GFP plasmid) to exponential phase, 0.154 ul of solution (measured)
  2. spot one 0.2 ul drop, on each agarose block/corresp. slide position, wait requisite time for each

Varying

  • wait time
    • 1 min, 5 min, 10 min

Deposition Protocol

  1. place on glass slide: (try this first)
    1. dry cells for ~0, 5, 10 min in middle of glass slide (also drying agarose for same time)
  • 2 replicates per wait time
  • 6 glass slides total
  • cover top of agarose w/ cover slip to simulate similar pressure in device
Time To do
min 0 pour agarose in 6 molds (108 ul)
min 20 remove agarose, place on square coverslips face up (using 2 blades)
min 20 place cells on 6 slides
min ~21 place agarose & slip on G1.1, G1.2,
min 25 place agarose & slip on G5.1, G5.2
min 30 place agarose & slip on G10.1, G10.2
  1. place on agarose: (try this after if time allows)
    1. after waiting 20 min for agarose to firm in mold, place on cover slip face up
    2. wait 0, 5, 10 min for agarose to dessicate
    3. apply cells to top surface of agarose
    4. gingerly move agarose to glass slide, face down


Measuring:

  • spot dispersion diameter

Data

dry time/surface 1 min 5 min 10 min
glass x x x
agarose x x x


Post-Mortem

Can't visualize cells easily and focus because agarose is drying too quickly, and becoming wrinkled Possible solutions:

  • decrease drying time (currently 20 min in mold, +1,5,10 min on cover slip)
  • dry/deposit cells in humid environment (this would take longer)