Biology Reference
In-Depth Information
Petri dish is on a flat and even surface, so the slice of final PDMS is flat
and even everywhere.
The PDMS spacers are reusable and should be good for a couple years.
When handling liquid PDMS wear gloves because it is greasy and hard to
wash out; but once cured, no gloves are needed.
Materials
Oven, Fisher Scientific* Isotemp* (13-247-637G, 3.75 cu. ft./106.2L).
PDMS, Sylgard, 184 SILICONE ELASTOMER KIT (Dow Corning).
Plastic cup/plastic fork.
Vacuum desiccator (Bel-Art Products, 08-594-15B, 260 mm diameter).
145
20 mm Petri dishes (Thermo Scientific, No. 240401).
24.1.2
Slide assembly
Figure 24.1
A shows a schematic of the final assembled slide. The PDMS slice is
strongly adherent to glass, and therefore, no glue or sealing agent is needed during
the assembly process:
1.
Prepare melted fission yeast media infused agarose, for example, YE5S 2%
agarose at 100
C.
2.
Wash the PDMS slice with 70% ethanol. (The PDMS slices are reusable, wash
before and after experimental use.)
3.
Dry the PDMS slice on KimWipes or paper towel.
4.
Place the PDMS slice on top of a glass slide. PDMSwill naturally adhere to the glass.
5.
Fill up the square well completely with the melted YE5S agarose, until it forms a
slight convex cap. While the agarose is still warm, immediately place a glass
coverslip on top.
6.
Gently, press down on the coverslip around its edges (never press down on top
of the well itself). This act to flatten out the agarose surface.
7.
Wait for
1 min for agarose to solidify. Then carefully remove the glass
coverslip from the well. (Excess agarose outside the well should also be
removed by scrape away.)
8.
Wait for
1 min further for the opened agarose well to dry.
9.
During this time, concentrate cells by centrifugation and then resuspend cell in
L.
10.
Apply 1
20
m
L of cell suspension on top of the agarose well.
11.
Place a glass coverslip on top gently at a small angle to make sure cells are
spread as evenly as possible on the agarose surface. The coverslip will adhere to
the PDMS spacer.
m
Notes
On steps 5 and 11, be careful to not create air bubbles. Bubbles in the
agarose or in the cells suspension will cause unwanted drifts or
movements during imaging.
