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

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
 * 2) Not using silicon molds, only testing bacterial dispersion on agarose/glass slide.
 * 3) waiting 20 min for agarose to dry in PDMS molds, at room temp; in future, will try different drying temps/dessication?
 * 4) 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)
 * 2) 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


 * 1) place on agarose: (try this after if time allows)
 * 2) after waiting 20 min for agarose to firm in mold, place on cover slip face up
 * 3) wait 0, 5, 10 min for agarose to dessicate
 * 4) apply cells to top surface of agarose
 * 5) gingerly move agarose to glass slide, face down

Measuring:
 * spot dispersion diameter

Data

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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)