Biology Reference
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E
Fig. 2 Fire-polishing pipettes. Fire polishing the tip allows the user to narrow the tip opening to gain
the desired shape and resistance. (A) The pulled pipettes are placed horizontally in a 2D holder mounted
on the stage of an inverted microscope with a long working distance objective 40 . On the opposite
side of the stage, a 3D manipulator is placed with a small platinum wire loop on which a bead of pipette
glass has been melted. (B) The unpolished pipette tip is brought into the same plane of view as the glass
bead, but at opposite sides of the periphery. (C) When current is passed through the wire, the bead gets
hot and expands into the center of the field. The tip of the pipette is transiently manipulated closer to the
bead until the opening at the tip starts to close. (D) When the tip narrows and attains rounded edges, the
pipette is withdrawn and the heat is turned o
V
. (E) A finished, polished pipette tip.
Table III
Checklist for obtaining giga-ohm seals
Cells are healthy
CO 2 regulation is accurate
No trypsin for 24 h and no transfection reagents in past 12 h
Maintained in salt solution at room temperature for less than 15 min
Pipettes are functioning properly
Pipettes are stored in dust-free box for 4 h after polishing
Pipette solution filtered through 0.45 m m mesh
Tip filled separately by capillary action before back filling; no bubbles
Pipette holder gasket sized to glass and fits snugly (but do not over tighten)
Suction line intact and dry
No mechanical vibrations transmitted to tip (see Table II )
Solutions
Bath solution has more solutes than pipette solution
Neither solution has proteins or ATP
At least one of the solutions has
0.1 mM Ca 2 รพ
 
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