Sutter Instrument P-97, Operation Manuals

Page: 49 / 66

39
5. TROUBLESHOOTING
5.1 Controlling Pipette Tip Shapes
5.1.1 Problem: What type of glass should be used?
The type of glass and the wall ratio I.D. (inside diameter) to O.D. (outside diameter) are two
of the most important variables in controlling tip size. For example, using borosilicate glass
with an O.D. of 1.0mm and an I.D. of 0.50mm will give tips of 0.06 to 0.07mm as
demonstrated in Program 0. Using the same settings, borosilicate glass 1.0mm O.D. and
0.78mm I.D. will form tips of 0.1 to 0.12mm. Aluminosilicate glass with an O.D. of 1.0mm and
an I.D. of 0.68mm will form tips of 0.03 to 0.04mm again with the same settings.
In general, the thicker the wall in relation to the O.D. of the glass the finer the tip will be,
and the thinner the wall the larger the tip will be. Thin walled glass will give the best results
in most experiments as it will have the largest pore for a given tip size. This means it will
have a lower resistance and will allow for easier injection of solutions. However, in many
cases with small cells, the thin walled glass will not form tips fine enough to obtain good
penetrations. In this case, heavier walled glass must be used.
5.1.2 Problem: The resistance of pulled pipettes is too low. How can a higher-resistance
pipette be pulled?
The first point to note is that there is very little correlation between tip size and electrode
resistance when pulling pipettes under 0.3mm. Most of the resistance of a microelectrode is
in the shank of the electrode behind the tip. Electrode tips that are 0.1 mm in diameter can
vary in resistance from 20M
Ω to 1000M Ω depending on the length of the electrode and what
is used for the filling solution. If the same solution is used then resistance may give an
indication of how well the electrode will penetrate a cell as the electrode with the higher
resistance will probably have a longer shank and a smaller cone angle at the tip. This
combination will aid in the penetration of cells where the cell is not a surface cell.
5.1.3 Problem: Okay, but how can tips be made even smaller.
1. The first thing to try in most cases is to increase the HEAT value. This will generally
decrease the tip size but it will also give a longer shank. If the higher resistance is not a
problem, this is generally the best solution. Continuing to increase the HEAT, however, is
not the final answer as too high a HEAT can lead to larger tips. In general, with 1.0mm
O.D.X 0.5mm I.D. borosilicate glass the finest tips will be formed when the glass pulls in 5
to 7 seconds after starting the pull.
2. If the electrode is now too long and results in a resistance too high to pass the necessary
current, then the next step is to increase the pull strength. In general, a pull strength of
125 will give tips of less then 0.1mm. Increasing the pull to 250 will reduce tip size about
5-10%. We recommend a pull of about 150 in most cases.
3. The last major variable to adjust is the amount of cooling of the glass during the pull. If
in the case of 1.0mm O.D. X 0.5mm I.D. borosilicate glass the pull takes place in 5-7
seconds, the tip size will not change with a change in the cooling air. The only change will
be in the length of the shank. If however the HEAT is such that the pull takes place in
more then 8 seconds, decreasing the cooling may somewhat decrease the tip size. Cooling
P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.43 - DOM (20161118)