Manual

1. Ensure the power cord is connected. Switch on the puller by pressing MAINS. The pulling rod moves to the right to the "ready" position.
2. Select a program from Table 1. Here program no. 10 (P10) has been used. Table 1 shows the settings of all puller parameters for the 20 standard programs for various electrodes and glass types. These programs are already installed by the manufacturer, but may be modified at will. Check the width of the heating filament in the puller. Please note that the programs P00 - P13 use a 3-mm-wide heating filament, the programs P14 - P19 a 4.5-mm-wide filament. This, too, may be varied as desired. The 3-mm filament is installed by the manufacturer and spare filaments of both sizes are supplied in the spare-parts box (for changing the filament see section 6.3 in the User's Manual, "Replacing the heating filament").
3. Open the Perspex lid, insert a glass pipette blank (type GC150 T or GC150TF, taken from the box supplied) into the open glass holders so that it rests on the two bearing surfaces (see Fig. 4, 1).
4. Press P(A) (Fig. 1, 9) and enter the corresponding number of the program (here 1 0) on the numeric keypad (Fig. 1, 11). In the display 10 should appear, confirming that program no. 10 is now activated.
5. Deactivate the tip polishing process which would normally follow pulling. Press P(B) and then AD; a three-digit number (xyz) will appear in the display. Press 0 (zero) on the numeric keypad and "enter" by pressing E (the enter key). The display should now show 0yz. If you make a mistake, clear the display (press C) press the correct numbers (0 y z) and enter (press E) See also point 5 below.
6. Close the Perspex lid and press START. The glass holders close automatically and heating begins. The pipette blank will be elongated a little by the heat and the preliminary pull, after which it is centred. The pulling and centering is repeated before the final pull which separates the two pipettes. Both glass holders now open and the finished pipettes can be removed (see description of programs). This is an example of a program with a total of three pulls, the first two of which were concerned with the preliminary centering and elongation (two preliminary pull program, see Table 1, note 2).

The puller may be used to polish patch electrodes after pulling. To activate the polishing process, it is necessary to alter one of the parameters stored in the puller before starting the pulling. This is done simply as follows: Press P(B) and AD. The number 0yz should appear in the display (see point 4 above). Change this by pressing 2 and then E (= enter) on the numeric keypad. The display should now show 2yz. If you make a mistake clear the display and enter the correct number (C 2 y z E) via the numeric keypad. Entering the value 2 has changed 0yz into 2yz. This indicates a polishing duration of 2 s at a heating current of yz0 (see Table 1, note 4). Two ranges (low and normal) of heating strength for the polishing process are available (see Table 1, note 3 and section 2.2, the description of the controls on the left-hand side of the control panel, in the User's Manual).

To pull and polish electrodes choose the same type of glass blank as before (GC150 T or GC150 TF).

1. Press READY , insert the glass blank into the glass holders, close the Perspex lid and press START. In this case the glass holders now do not open after pulling.
2. Press START again. The right-hand holder moves automatically a little to the right so that when the heating filament subsequently moves into outside position it encloses the tip in the middle of its curved surface. The heater now moves to the outer position and heat-polishes the right-hand pipette. When this is concluded the right-hand glass holder will open automatically, allowing the polished pipette to be removed.
3. Press START yet again. The left-hand pipette moves into the polishing position and is heat polished, after which the holder opens and the pipette can be removed.

The polishing parameters can be altered before pulling by using the keys P(A), t(H) and s(H) (see Table 1, notes 5 and 6 and description of program 10). Values between 001 and 090 are recommended. The values actually set can be displayed by pressing P(A) and then t(H) or s(H) and changed when the puller is in the "ready" condition. The polishing parameters cannot be changed after starting the pulling/polishing process. Once started, the polishing process can be aborted only by switching off the puller. To deactivate the polishing process in a program press, before starting the pulling program, P(A) and AD (the display should show 2yz, see above) and then 0 and E on the numeric keypad (the display should now show 0yz). If you make a mistake press C (clear), 0 y z E (enter) on the numeric keypad (see also point 4 above).

The two groups of controls are located on the sloping top panel of the instrument. This panel is hinged (Fig.1, 1 and 2) to allow service access. On the right hand side is the group of illuminated switches and indicators controlling individual pulls, on the left are keys defining the variables and a numeric keypad. These are used to select (and alter if necessary) the 80 stored programs and the time sequence of the pulls.

2.1 Right control panel

The puller is switched on and off using the MAINS switch (3). The lower keys bring the puller into a state of readiness (READY, 4), start a pull ( START, 5) or, in case of trouble, interrupt the pull sequence ( BREAK, 6). The latter lights up when the program detects a disturbance in the pulling sequence which stops the program. The stages of the pulling sequence are indicated by the real time counter (7, 100-ms units from the start of the pull) and the lamps (8) which light up at various critical points within the sequence: "F(TH)" is lit as long as the heater is on and a small sensing or preliminary or sensing pull is operating. It is extinguished when a (programmable) threshold extension is reached. At this point "(TH)" lights up. "F1" is lit when the first phase of the main pull begins, "F2" lights up when the second phase of the main pull begins.

Pressing the keys P(A) or P(B) (9) causes the number (e.g. P14) of the last-used program to appear in the display (10). To change the program selected simply type the appropriate number (e.g. 0 9) on the numerical keypad (11 ), it will appear in the display (10).

Note: The programs P00, P09, P10, P11, P12, and P13 have been tested in each individual puller and the values of the parameters stored in the memory adjusted to achieve the specified tip characteristics (see tables 1 and 2). The numeric values may thus deviate slightly from the values both in the table and in any numeric examples given in the manual.

The keys P(A) and P(B) (9) further define the effects of pressing the function keys (12, see also section 3, Description of programs). Depressing a function key ( 12) causes the value set for this variable in the current program to appear in the display (10). If the value of this variable is to be changed, simply type the new numerals (from left to right) on the numeric keypad (11) and press E (enter). The new value will appear in the display (10 ). For example, to change the value 214 to 014 press 0 and E. If a mistake is made, clear the display (C) and enter the whole new number (the entire sequence would then be C 0 1 4 E).

To obtain single-pull programs press only the program key P(A) (Fig. 1, 9) and enter the number of the programm (P00 - P79) on the numeric keypad (11 ). Do not press P(B). AD must have the value 000. The various functions (12) now apply to a single pull.

Single-pull programs proceed with the following sequence:

On switching the puller on (MAINS, Fig.1, 3) the pulling rod with the left-hand glass holder (Fig.1, 16) moves into the "ready" position and the READY switch lights up.

On pressing START the glass holders close automatically and clamp the glass. The timer (Fig.1, 7) is activated. The heating current is switched on (power H) and the filament moved into position. The force coil exerts a small sensing pull with force F(TH) (a typical value for F(TH) would be 020). When heated sufficiently the glass under the filament becomes plastic and the traction on the pulling rod causes the glass to elongate. The pulling rod thus moves back. The distance moved is measured and the sensing pull switched off when the set distance threshold [ s(TH)] is reached. This sensing pull routine serves to compensate for small differences in pipette dimensions.

At the conclusion of this sensing pull, the main pull (phase 1) is initiated after a delay [t(F1)], with a force F1 . This results in further elongation and, on reaching the distance s(F2) the pulling force changes to F2. Within this pulling sequence heat application is terminated in either of two ways:

• If Delay Heatstop t(H) has been selected (Distance Heatstop s(H) must be 000) the heating continues for the time t(H).
• If Distance Heatstop s(H) has been selected (Delay Heatstop t(H) must be 000), heating continues until the distance s(H) is reached.

After reaching t(H) or s(H) the heat is switched off and the heater retracted. The pull sequence pulls the pipettes apart, the sequence stops, the glass holders open and the pipettes may be removed.

These programs include up to nine (9) preliminary pulls and produce steeply tapering electrodes or patch-clamp electrodes. Press either P(A) or P(B) and enter the program number (P00 - P79) on the numeric keypad. In these programs the variables for the preliminary and main pulls are selected by first activating P(A) or P(B).

After pressing P(A), the functions H, F(TH) and s(TH) define the characteristics of the first pulling stages (one, two, three, or more preliminary pulls). There are two ranges of heating strength available for the main pull, a normal and a low range. The key AD in combination with P(A) selects the range. The low range allows the control of heating strength to be more precise and is particularly suited to the production of patch electrodes with large tip diameters (see section 2.2 and Table 1, note 1). The key AD also determines whether the first stage of the pulling sequence employs one, two, three, or more preliminary pulls (see description of the function keys above and Table 1, note 2) . The remaining functions are normally disregarded (except for t(H) and s(H), see section 3.3, Polishing patch electrodes).

After pressing P(B) , the functions keys define the variables for the main pull. AD defines whether or not electrode tips are polished (see section 2.2, function AD, section 3.3 Polishing patch electrodes and Table 1, note 4).

The first phase of a multi-pull program is initiated and proceeds as in the single-pull programs (section 3.1), except one, two, three or more preliminary pulls are employed. The combination of the values set for the variables is such that a constriction is produced in the glass blank, but the two halves are not pulled apart. At the end of the first preliminary pull the glass is moved to the right, so that the constriction is aligned with the heating filament. This can be repeated a second or a third time (up to nine times). The final pulling phase (the main pull) then commences automatically and proceeds exactly as in the single-pull program.

Pressing AD after P(B) allows selection of a fully automatic polishing routine after completion of the final pull. With this program activated, the glass holders do not open after the final pull. After pressing START, the right-hand holder moves to the right and the heater is switched on for the selected time at the selected power (see section 2.2, function key AD and Table 1 note 4)??? . After the heater is switched off the glass holder opens, allowing the finished electrode to be removed. Pressing START again repeats the process for the left-hand electrode. The distances moved by the glass holders to the polishing positions are set using the keys t(H) (right-hand electrode) and s(H) (left-hand electrode) when P(A) is activated. Values between 001 and 080 are suggested (see Table 1, note 4).

Table 1 summarises programs which have been most widely used. Typical uses of the electrodes/pipettes are also given for orientation (see also First steps). Despite the utmost care taken in the construction small variations between individual pullers are unavoidable thus requiring minor adjustments to the values given, in particular Heat (strength), the Heatstop variables and Delay Pull 1. Descriptions of programs P10 - P13 with commentaries for orientation are found on chapter 5.1 to 5.4.

Table 2 summarises qualitatively the effects of changing individual parameters on the shape of the electrode/pipette.

The following is a step-by-step description of a pull, using program P12 (values as in Table 2, and Diagram 1 and 2).

1. When START is pressed, the glass holders close, the heater advances, is switched on and heats the glass (heating strength H = 600, Diagram 1 A and Figure 1). The preliminary pull (low force, (F(TH) = 040, diagram 1B t0, Fig. 1) starts to extend the softened glass until a certain distance threshold (s(TH) = 030 diagram 1C, t1 -t2, Fig. 1) is reached. At this time (t2) the heater (Diagram 1A) and the force F(TH) are switched off and the glass tubing is moved to the right to bring the constriction in the glass to the position of the heater (Diagram 1, t2 - t3).
2. This preliminary pull can be repeated once or twice more (i.e., a maximum of three times altogether). For the second (and third, if chosen) preliminary pull the program uses the same variables, i.e., heat 600, 040 for the force and 030 for the elongation distance threshold.
3. After concluding the preliminary pull(s), the program continues with the final pull (P(B), Diagram 1, t5 and Fig. 2) . After the pipette has been repositioned to the right, the heater is advanced and heats the glass again (heat strength H = 650 Table 1, row P(B) program P12 and Fig. 2). A small force (the sensing pull) is applied (F(TH) = 020) such that when the glass has softened it elongates until the distance threshold s(TH) = 015 is reached (Diagram 1C, t6) In this phase of the pull the stiffness of the glass is sensed. Reaching this point results in the force F(TH) being switched off, thus terminating the elongation. The two programmed delays (t(H) = 100 and t(F1) = 130 now become operative. After delay t(H) the heat is switched off (Diagram 1, t7) and the glass cools a little. After delay t(F1) the first main pulling force F1 = 400 is switched on (Diagram 1 , t8), which elongates the glass to a further relative distance threshold s(F2) = 005 at which point the second main pull F2 = 700 is switched on (Diagram 1 t9) and finally pulls the pipettes apart.

The final pull is also shown magnified on diagram 2.

Pipette type: Patch
Glass: Harvard Apparatus GC150TF thinwall or GC150F thickwall
Tip size: 2.0 µm

To achieve smaller tip diameters decrease P(B) t(F1) or, less preferable, increase P(B) H.

Pipette type: Patch
Glass: Harvard Apparatus GC150TF thinwall or GC150F thickwall
Tip size: 0.8 µm

To achieve larger tip diameters decrease P(B) H or decrease P(B) t(F1) or decrease P(B) F2.

Pipette type: Intracellular
Glass: Harvard Apparatus GC150F thickwall
Resistance: 20-40 MOhm

To achieve electrodes with lower tip resistance decrease P(B) H or increase P(B) t(F1) or decrease P(B) F2.

Pipette type: Macro-Patch
Glass: Assistent Haematokrit No. 564
Tip size: 28 µm

The high value of P(B) s(TH) (045) ensures that the pipettes are already separated, so F1 and F2 have no influence.

To achieve smaller tip diameters increase P(B) H or decrease P(B) F(TH).

The following explanations are intended as an aid to achieving more rapidly the desired results with the DMZ Universal Puller. It must be understood that the various parameters can influence each other mutually, so that in making tests, it is advisable to alter only one parameter at a time. To understand better the processes involved in pulling and the explanations given below, the reader should also consult the diagrams.

1. Increasing (↑) the heating H raises the temperature of the glass during the sensing pull F(TH), the delay periods and the main pulls F1 and F2. The result is a smaller (↓) tip diameter and a less steep taper. Excessive heat results in extremely long tips. Conversely, reducing the heat prolongs the pulling process resulting in very short tips. In the extreme case, the glass may even tear apart or the pulling process be terminated.
2. The sensing pull F(TH) begins immediately on switching on the heating. This is a very weak pull which only begins to move the pulling rod after the glass has reached its melting point. Increasing (↑) the sensing pull (max.: 100) results in the pulling rod reaching its threshold s(TH), at which the main pull begins, sooner. This is equivalent to reducing the duration of heating for the duration of the sensing pull so that the glass at the melting site has less time to become fluid than with a lower sensing pull (min.: 020) and is thus more viscous, resulting in greater (↑) tip diameters.
3. The distance s(TH) is the threshold which terminates the sensing pull F(TH). Increasing (↑) this threshold (max. 127) means that the pulling rod takes longer to reach this point, thus increasing the duration of the sensing pull. The glass thus not only remains longer in the position where it is melting, thus becoming more fluid (see 2. above), it also thins out, and the tip diameters decrease (↓). An high value for s(TH) may result in the glass separating before s(TH) is even reached. In this case all subsequent parameters have no further influence on the tip form. An example of this special case is found in Program P13 (Table 1).
4. At the instant at which the pulling rod reaches s(TH) and the sensing pull f(TH) terminates, two variable delays begin: delay heatstop t(H) and the pull delay t(F1). The prerequisite for these is that t(H) has been selected (see manual). During delay t(H), the heating continues at the same intensity.
   
   
   1. When delay heatstop expires, the heating is switched off and the heating unit retracts . The longer (↑) the time selected for t(H), the longer heating continues and the smaller (↓) the tip diameter. For t(H) to be effective, the distance heatstop s(H) must be set to zero (000) (see 7 below).
   2. The heatstop distance s(H) can be used as a (mutually exclusive) alternative to delay heatstop (see 4a above). This possibility is described in greater detail below (see 7 below).
   3. The pull delay t(F1) begins at the same time as the delay heatstop (see 4, 4a above). In general, the value for t(F1) should be greater than that for t(H), so that the pull F1 starts after the heating phase has terminated and the glass is beginning to cool. (See diagrams on pages 14 and 15). Increasing t(F1) (↑) results in the glass being cooler when the pull F1 starts and tip diameter is larger (↑). Varying t(F1) allows precise control of glass temperature from the end of the heating phase into the cooling phase, without changing the global heat setting and is thus the preferable means of varying electrode tip diameter.
   
   
5. Raising (↑) the force of the main pulls F1 and F2 increases the speed with which the glass is pulled apart in the cooling phase. The reduced cooling time results in smaller (↓) tip diameters.
6. The threshold distance s(F2) , upon reaching which the second main pull F2 is initiated, has the following effect: F1 extends the glass until, on reaching s(F2), the second pull F2 starts. If F1 is relatively small compared with F2 the higher the value for s(F2) the cooler the glass when F2 starts: increasing (↑) s(F2) increases (↑) tip diameters (providing, of course, that the glass has not already separated before reaching s(F2), in which case F2 has no relevance).
7. The heatstop distance s(H) is a (mutually exclusive) alternative to delay heatstop t(H) (see 4 above). To use this, s(H) can be given values of 001 - 127; delay heatstop t(H) must be set to zero (000). Under these circumstances, heating continues during F1 and F2 until a certain extension (the threshold s(H)) of the glass is reached, upon which the heating is turned off and retracted. Increasing(↑) s(H) thus prolongs heating during the main pulls F1 and F2 resulting in long tips with small (↓) diameters.