Glass capillaries (GD-1, NARISHIGE) were pulled by a puller (PC-100, NARISHIGE); the temperatures were set at 66.2 and 62.0 °C. Our microinjection system consists of an inverted microscope (AXIO Vert. A1, Zeiss) equipped with an injector (IM-31, NARISHIGE) and manipulators (MMN-1 and MHW-103, NARISHIGE). Tardigrades were mounted as described previously by Tenlen et al. for RNAi experiments without anesthesia (Fig. 1B ) (54 (link), 55 (link)). After the microinjections, individuals were collected and transferred to a cuvette (CUY505P5, NEPA GENE) for electroporation using a super electroporator (NEPA21 type 2, NEPA GENE). The poring pulse was emitted twice at 250 V for 5 ms with a 50-ms interval, and the transfer pulse was emitted five times at 30 V for 50 ms with a 50-ms interval. Tardigrades were maintained with algal food on an agar plate until observation at 17 °C.
Mmn 1
Manufactured by Narishige
Sourced in Japan
The MMN-1 is a micromanipulator produced by Narishige. It is designed for precise and delicate positioning of tools or probes for various experimental applications. The MMN-1 provides micrometer-level control of movement in three dimensions.
Automatically generated - may contain errors
2 protocols using mmn 1
1
Microinjection and Electroporation of Tardigrades
2
Polystyrene Tube Bilayer Formation
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A cut polystyrene tube (internal diameter, 6 mm) glued to the coverglass as a bottom plate was used as the bath chamber to measure the channel currents. An aqueous recording solution (250 μl) was poured into the bath chamber. A lipid solution was prepared by dissolving 1,2-diphytanoyl-sn-glycero-3phosphocholine (Avanti Polar Lipids, Alabaster, US) in n-decane to 20 mg/ml and then approximately 100 μl of the lipid solution was layered over the recording solution layer. For the bilayer formation, it is only required that the recording solution is covered with the lipid solution. It is not needed to precisely control the amount of lipid solution.
Bilayers were made by moving the gold electrode through the lipid solution layer into the recording solution. The depth the tip was dipped into the solution was usually 10 μm -1 mm. Precise determination of the depth was not necessary because we confirmed that coarse dipping using a coarse positioning manipulator (MMN-1, Narishige, Japan) was sufficient for bilayer formation. The experimental setup is shown in Fig. S1 (Supporting Information).
Bilayers were made by moving the gold electrode through the lipid solution layer into the recording solution. The depth the tip was dipped into the solution was usually 10 μm -1 mm. Precise determination of the depth was not necessary because we confirmed that coarse dipping using a coarse positioning manipulator (MMN-1, Narishige, Japan) was sufficient for bilayer formation. The experimental setup is shown in Fig. S1 (Supporting Information).
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