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History: Protocol adapted from Michael Koelle (08/23/94), edited by Erik Andersen (08/10/09), Troy Meikle (04/24/18), and Kathy Vaccaro (03/09/20).
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You will have a couple of frustrating sessions when you first attempt this technique. Everyone seems to master injection after a few days, and it works quickly and reliably once you have some experience.
Make a lot of these at a time; they last forever.
Using a transfer pipette, drop melted 2% agarose in sterile water on a 24x50 mm coverslip. The size of the droplet should be moderate — not too large, not too small — though this isn't precise nor a rigid requirement.
Drop a second coverslip on top, which will flatten the agarose into a thin pad. Try to avoid air bubbles, but a few won't hurt anything.
When the agarose has hardened (more than 5 seconds), slide off the top coverslip.
Breathe on the coverslip to see which side the pad stuck to. Use this top coverslip as the bottom coverslip for the next pad — its thin coating of agarose will make the pad stick to it instead of the fresh top coverslip.
Place the coverslips flat in a box and cover the box with aluminum foil to dry. The box can be left out on the bench overnight, baked in an oven at 65°C for 1 hour, or baked at 80°C for 15 minutes.
Once dried, store the pads in the original coverslip box.
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This can be made into an assembly line by lining up many coverslips on the edge of a bench, so you can quickly pick them up, with an equal number of coverslips perpendicular to the first set behind them. Spot the perpendicular coverslip, pick up the one at the edge of the bench, and drop it on top. Repeat on down the line. You can make 200 coverslips in about half an hour.
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We use "Glass 1BBL w/FIL 1.0 mm 4 IN" filaments (#1B100F-4) from World Precision Instruments. Keep these clean and always immediately recap the tube after removing a filament.
We use a Sutter Instruments model P-87 needle puller.