Pipette Cookbook 2018 P-97 & P-1000 Micropipette Pullers Rev. F The Pipette Cookbook was written, compiled and created by Adair Oesterle with Sutter Instrument Company. A printed version of the Pipette Cookbook is included with all new P-97 and P-1000 Pullers and the pdf can found and downloaded for free through the Sutter web site. http://www.sutter.com/contact/faqs/pipette_cookbook.pdf Revised and expanded versions of the Pipette Cookbook are released every two to three years. Please contact Sutter if you have any recommendations, comments and/or corrections to offer. We welcome your feedback! Adair Oesterle at the Getty Museum – not pulling pipettes! Sutter Instrument Company One Digital Drive Novato, CA 94949 415-883-0128 www.sutter.com 2 TABLE OF CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 5-6 Using the Pipette Cookbook with the P-87, P-80/PC & P-1000 . . . . . . . . . . . . . . . . . . . Pg. 6 Sutter Capillary Glass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 7 Sutter Filaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 8 Ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 9 Parameter Settings Defined . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 10 General Guideline for Parameter Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 11 Ramp Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 12 Installing a Filament - Step-by-Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 13-18 Centering Filament Over Air Jet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 17-18 Chapter 1 Electrophysiology Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 19 Extracellular Recording Electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 20 Intracellular Recording Electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 21-22 Additional Concerns about Intracellular Recording . . . . . . . . . . . . . . . . Pg. 23-26 Patch Pipettes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 27-34 Pre-Heat Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 29 Writing a Stable Patch Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 30-32 Cheet Sheet – Parameter Adjustment Guide . . . . . . . . . . . . . . . . . . . . . . Pg. 33 Chapter 2 Adherent Cell, C.elegans, & Drosophila. . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 35-36 XenoWorks Digital Injector Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 37-38 Pre-Pulled Injection Pipettes – Do You Know What You Are Buying? . . . . Pg. 39-40 Zebrafish Embryo Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 41-42 Chapter 3 Bee-Stinger Needle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 43-44 Chapter 4 Pronuclear Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 45-48 Chapter 5 Embryonic Stem Cell Injection (ES Cell) . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 49-50 Chapter 6 Intracytoplasmic Sperm Injection (ICSI) . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 51-52 Chapter 7 Nuclear Transfer (NT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 53-54 Chapter 8 Holding Pipettes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 55-56 Chapter 9 Xenopus Oocyte Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 57-58 3 Chapter 10 Large Pipettes, 20µ to 200µ Tips, Methods on Breaking Back Tip, Glass-on-Glass Technique & Using the Ceramic Tile (CTS) . . . . . . . . . . Pg. 59-62 Chapter 11 Aspiration Pipettes: Cell Selection, Cell Transfer, Single Cell Mechanics & Blastomere Biopsy. . . . . . . . . . . . . . . . . . . . . . Pg. 63-64 Chapter 12 Aluminosilicate Glass and Custom Programs. . . . . . . . . . . . . . . . . . . . . . Pg. 65-68 Chapter 13 ZIKA Research and Mosquito Egg Injections . . . . . . . . . . . . . . . . . . . . . Pg. 69-73 Chapter 14 Crazy Lab Lore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 75-81 Chapter 15 Variability and “The 15 Questions”. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 83-84 Chapter 16 Installation of Box or Trough Filament - Review . . . . . . . . . . . . . . . . . . . Pg. 85-87 Eccentric Adjustments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 88 Chapter 17 Filamented Glass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 89 Chapter 18 XenoWorks Microinjection System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 91-93 Chapter 19 BV-10 Beveler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 95-96 GENERAL LOOK UP TABLES Type A, B, C, D, & E Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . Pg. 97 General Look Up Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 98-105 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pg. 106-108 P-97 Pipette Puller *New* P-1000 Pipette Puller * The New P-1000 Pipette Puller has the “Pipette Cookbook” programs incorporated into its memory. The new Cookbook feature for the P-1000 can be used to look up and install a majority of the programs found in this text. By selecting the filament type, glass dimension, and application you can install a pre-written program. To fine-tune the program you installed from the cookbook directory, please refer to pg 11 for instructions on how to adjust the parameter settings. 4 Introduction The Sutter Instrument Pipette Cookbook is organized according to application and describes the required pipette needed for that application. While we have attempted to cover a wide range of applications and types of pipettes, it is possible we have yet to include what you might be looking for. Additional applications are occasionally added to new revisions of the cookbook and if you have suggestions for future revisions, please contact Sutter Instrument Company. At the beginning of each chapter you will find a general discussion of each application and a detailed description of the morphology of the pipette (taper length, tips size, resistance, etc) needed for your application. In each chapter we provide the “ideal” combination of glass size (outer and inner diameters) and filament type for your application, along with the recommended parameter settings with which to start. We then provide suggestions on how to adjust and “tweak” the recommended parameter settings in case you need to modify the morphology of the resulting pipette. When applicable, we have also provided information on the XenoWorks microinjection equipment, the set up and the settings that would be required for that application. The “ideal” filament and glass combination, and the associated recommended parameter settings we have provided in each chapter have been established over years of experience, and are a result of in-depth research results and customer feedback. If you are unable to match the filament and glass combination that is provided for a specific application or you lack the “recommended ingredients,” you can refer to the “General Look Up Tables” to find an alternate program. The General Look Up Tables are organized according to the type of filament installed in your puller and the dimension of glass (OD and ID). Programs are listed in the Tables as Type A, B, C, D, and E. Each “Type” classification is explained on page 97. It is important to keep in mind that what is provided in the General Look Up Tables might not be “ideal” for your application. There are some combinations of filament and glass that do not work well for a given application and can create very unstable results. So, we would like to emphasize again that what has been provided in each chapter is the best approach we have come to recognize. The most common sources of difficulty in producing the right shape of pipette can be attributed to the use of poor parameter settings. Just one poorly adjusted parameter setting in the program - too high or too low - can lead to a lot of variability in tip size, taper length and resistance. These very high or low settings can be seen as “bad ingredients” in your recipe. For a general guideline on the parameter settings and the range of values we recommend, please refer to page 11. If you stay within the suggested range for each parameter, it will be less likely that you will “get lost” or off track when trying to write a program or adjust your settings. It is possible that the not-so-ideal parameter settings were established in a somewhat haphazard manner by various well-intentioned researchers who are possibly no longer in the lab. It is also conceivable that the program one is using had been adopted from someone who had a different model of Sutter puller and/or a different combination of filament and glass. Please be aware that simply using a program recommended by someone else can often lead to undesirable tip sizes, and in worse cases, the unfortunate event of burning out your filament. If you have acquired your settings from someone outside of Sutter, it is best to run a ramp test to make sure the heat settings you have been advised to use will not damage or burn out your filament. Information about the Ramp Test can be found on page 12. 5 If a program in this Cookbook does not produce the proper pipette, this might be a result of a poor alignment or mechanical adjustment on your puller. It is important to make sure your puller is in good working order. Be sure to check that the filament is perfectly centered over the air jet, the air jet is set 2 to 3mm below the base of the filament, the glass capillary is properly positioned within the filament, and the filament shape is correct. If you find that the program provided and the suggested changes to the parameter settings do not produce good results, please refer to page 11, “General Guideline for Parameter Settings” and Chapter 15, “Variability.” If you feel your puller might be in need of a tune up or repair, please contact Sutter Instrument and inquire about having your puller refurbished. Using the Pipette Cookbook with a P-87 or a P-80/PC To adapt the P-97/P-1000 Pipette Cookbook programs to an earlier model Sutter Micropipette Puller, including the P-87 and P-80/PC, please do the following: § If you have a P- 87, reduce the heat setting listed in the chart by 5-10 units. The power available to the filament in the P-97 is 25% higher than the P-87 and the rate of current increase during a ramp test on the P-97 is more gradual. These differences require one to alter the heat setting when using a P-97 program on a P-87. If a Delay Value is provided, you will need to install a Time Value between 175 and 250 in place of the Delay Value. § If you have a P-80/PC, reduce the heat setting listed in the table by 10 units. The Time Mode on the P-80/PC functions just like the Delay Mode on the P-97 and P-1000. If a Delay Value is provided in Pipette Cookbook program, install this same number as the Time Value on your P-80/PC. If a Time Value is provided in the Pipette Cookbook program, please install a Time Value between 50 to 110 on the P-80/PC. § The Pressure of the out-going regulator of the P-80/PC should be set to 50psi. The airflow of the Nitrogen is controlled by adjusting the valve opening of the air solenoid, which is a vertical micrometer dial behind the left puller bar. Micrometer settings between 1 and 1.25 are recommended. *For detailed information about the cooling modes, Time & Delay, please refer to your P-97 or P-1000 manual. Using the Pipette Cookbook with a P-1000 The New P-1000 Pipette Puller has the “Pipette Cookbook” programs incorporated into its memory. The new Cookbook feature for the P-1000 can be used to look up and install a majority of the programs found in this text. By selecting the filament type, glass dimension, and application you can find and install a pre-written program. To fine tune your program, please refer to page 11 for instructions on how to adjust the parameter settings. Aluminosilicate programs and Type E programs were not incorporated into the P-1000. Programs for pulling Aluminosilicate glass can be found in Chapter 12. You might also want to try using the new Pre-Heat Feature on your P-1000 puller to help stabilize your programs. For additional advice, please contact Sutter Technical Support. 6 SUTTER CAPILLARY GLASS Standard Wall Borosilicate Tubing (With Filament) Catalog Number Outside Diameter Inside Diameter Overall Length Pieces per Package BF100-50-10 1.00mm 0.50mm 10cm 225 BF100-50-15 1.00mm 0.50mm 15cm 225 BF100-58-10 1.00mm 0.58mm 10cm 250 BF100-58-15 1.00mm 0.58mm 15cm 250 BF120-60-10 1.20mm 0.60mm 10cm 225 BF120-69-10 1.20mm 0.69mm 10cm 250 BF120-69-15 1.20mm 0.69mm 15cm 250 BF150-75-10 1.50mm 0.75mm 10cm 225 BF150-86-7.5 1.50mm 0.86mm 7.5cm 250 BF150-86-10 1.50mm 0.86mm 10cm 250 BF150-86-15 1.50mm 0.86mm 15cm 250 BF200-100-10 2.00mm 1.00mm 10cm 225 BF200-116-10 2.00mm 1.16mm 10cm 250 BF200-116-15 2.00mm 1.16mm 15cm 250 Standard Wall Borosilicate Tubing (No Filament) Catalog Number Outside Diameter Inside Diameter Overall Length Pieces per Package B100-50-10 1.00mm 0.50mm 10cm 225 B100-50-15 1.00mm 0.50mm 15cm 225 B100-58-10 1.00mm 0.58mm 10cm 250 B100-58-15 1.00mm 0.58mm 15cm 250 B120-69-10 1.20mm 0.69mm 10cm 250 B120-69-15 1.20mm 0.69mm 15cm 250 B150-86-7.5 1.50mm 0.86mm 7.5cm 250 B150-86-10 1.50mm 0.86mm 10cm 250 B150-86-15 1.50mm 0.86mm 15cm 250 B200-116-10 2.00mm 1.16mm 10cm 250 B200-116-15 2.00mm 1.16mm 15cm 250 Thin Wall Borosilicate Tubing (With Filament) Catalog Number Outside Diameter Inside Diameter Overall Length Pieces per Package BF100-78-10 1.00mm 0.78mm 10cm 250 BF100-78-15 1.00mm 0.78mm 15cm 250 BF120-94-10 1.20mm 0.94mm 10cm 250 BF120-94-15 1.20mm 0.94mm 15cm 250 BF150-110-7.5 1.50mm 1.10mm 7.5cm 250 BF150-110-10 1.50mm 1.10mm 10cm 225 BF150-117-10 1.50mm 1.17mm 10cm 250 BF150-117-15 1.50mm 1.17mm 15cm 100 BF165-120-10 1.65mm 1.20mm 10cm 250 BF200-156-10 2.00mm 1.56mm 10cm 250 BF200-156-15 2.00mm 1.56mm 15cm 100 Thin Wall Borosilicate Tubing (No Filament) Catalog Number Outside Diameter Inside Diameter Overall Length Pieces per Package B100-75-10 1.00mm 0.75mm 10cm 225 B100-75-15 1.00mm 0.75mm 15cm 225 B120-90-10 1.20mm 0.90mm 10cm 225 B150-110-7.5 1.50mm 1.10mm 7.5cm 225 B150-110-10 1.50mm 1.10mm 10cm 225 For a list of our Aluminosilicate & Multibarrel Borosilicate Glass, please refer to our web site (www.sutter.com) 7 Filaments & Accessories Box Filaments P-1000, P-97, P-87, P80PC, P80C, PC-84, P-77B FB215B 2.0mm square box filament, 1.5mm wide…………………………………. FB220B 2.0mm square box filament, 2.0mm wide…………………………………. FB230B 2.0mm square box filament, 3.0mm wide…………………………………. FB255B 2.5mm square box filament, 2.5mm wide…………………………………. FB245B 2.5mm square box filament, 4.5mm wide…………………………………. FB315B 3.0mm square box filament, 1.5mm wide…………………………………. FB320B 3.0mm square box filament, 2.0mm wide…………………………………. FB330B 3.0mm square box filament, 3.0mm wide…………………………………. Trough Filaments P-1000, P-97, P-87, P80PC, P80C, PC-84, P-77B FT315B 1.5mm wide trough filament………………………………………………. FT320B 2.0mm wide trough filament………………………………………………. FT330B 3.0mm wide trough filament……………………………………………..... FT345B 4.5mm wide trough filament……………………………………………..... Accessories BX-10: Pipette Storage Box (Holds 10 pipettes)……………………………………. BX-20: Pipette Storage Box (Holds 20 pipettes)……………………………………. Custom Filament: Custom platinum/iridium filament……………………………... FPS Fire polishing spacer for P97, P87, and P2000 pullers…………………………. FS1875 Platinum/iridium sheet, 18mm x 75mm x 0.05mm (0.002in)…………….... CTS Ceramic tile for scoring glass (large tips 20 to 200 microns)………………….. IMOXAB Instruction manual (specify product when ordering)…………………….. Pipette Storage Boxes Sutter Capillary Glass Ceramic Tiles (See Chapter 10) Item# BX10 or BX20 Page 7 for Parts Numbers Item # CTS 8 THREE MAIN INGREDIENTS: • GLASS • FILAMENT TYPE & SIZE (Heating Element) 2.5mm x 2.5mm Box Filament Box Filament Centered Over Air Jet • PARAMETER SETTINGS = PROGRAM HEAT PULL VEL DEL PRESSURE Ramp +10 60-90 80 90 200 GLASS Within each Chapter describing a specific application, Slice Patch Recording for example, a specific glass type, a specific wall thickness, and filamented or non-filamented glass is recommended. Quartz glass is not discussed much in this cookbook because the filament-based pullers like the P-97 and P-1000 cannot melt Quartz glass, but it is mentioned (and recommended if you have a P-2000 Puller) in Chapter 13 – Zika Research and Mosquito Egg Injections. More information on the glass can be found in Chapter 14– Crazy Lab Lore. FILAMENTS The filament in your puller is the heating element used to melt the glass. The filaments come in two basic shapes, box or trough. See page 8 for list of filaments. They also come in various widths and IDs (inner diameter or opening of the box or trough). The filament is made of Platinum:Iridium (90:10) and the proper heat setting to use for your filament is determined by running a RAMP TEST. The ramp test is further discussed on page 12. 9 PARAMETER SETTINGS The P-97 and P-1000 puller use the following parameters to control the melting and pulling of the glass: Each application described in the Pipette Cookbook will offer a starting program where the heat, pull, velocity, time/delay and pressure settings are suggested. Please see the SutterInstrument YouTube Channel to find the Scientists Empowering Scientists Webinar, “How to Make Better Pipettes”, which describes the parameters settings in more depth. The manual that came with your puller will also have a description of the parameter settings. A brief description of each setting is discussed below: HEAT – This is the amount of current supplied to the filament. The value does not represent the temperature, but indicates how much current is delivered to the filament. The filament needs to get hot enough to melt the glass, and this heat value is dependent on the filament shape, the filament size and the size and wall thickness of the glass. If you use a heat setting that is too high or too low, you risk burning out the filament or damaging the puller. So it is not a good idea to guess at the heat setting. You can also introduce a lot of variability if the heat setting is not ideal. To determine the proper and safe HEAT value to use, one should run a RAMP TEST. For more information about the Ramp TEST, see page 12. PULL – This is the hard pull introduced to the glass after the glass has softened. This pull value determines the amount of current supplied to the pull solenoid to create the hard pull. The puller can pull the glass out with a hard pull (Pull = < 0 >) where the puller only uses the gravitational weight of the plunger inside the puller to pull on the glass. A pull of < 0 > is typical when pulling a patch pipette where you want a 3-5mm short taper and a 1-3µm tip. A pull of 50-150 is typical when making a sharp electrode of 30 to100 MΩ or when making a microinjection needle. VELOCITY – This is the rate of separation of the puller bars when the glass first starts to melt. The velocity is detected by a transducer inside the puller, a patented feature, and the velocity has a direct correlation to the viscosity of the glass. The velocity is the “trip point” for turning off the heat and starting the cooling and the hard pull. When the velocity is low, the pipette taper will be shorter. When the velocity value is low enough, as when pulling a patch pipette, the puller will pull in multiple cycles or “LOOPS”. A velocity of 18 - 65 is typical when pulling a patch pipette, and when using a one-line program, the lower the velocity is, the more times the program will loop. LOOPING is further discussed on page 29. A pull of 50-150 is typical when making a sharp electrode of 30 to100 MΩ or when making a microinjection needle. TIME – This is the duration of air used to cool the glass and the filament as the glass is being pulled. When using the Time mode, the glass softens, the velocity trip point is reached, and then the glass is cooled at pulled simultaneously. The duration of cooling is determined by the value of the Time where each unit of time is equivalent to 0.5 milliseconds of cooling air. Traditionally a Time of 150 (75ms) is recommended when using a trough filament or when using thin walled glass and making a pipette for slice patch recording. A higher Time value of 250 (125ms) is recommended when using a box filament and pulling thick walled glass. DELAY – This is the alternative mode of cooling which provides a longer duration of cooling (300ms) than the Time mode (max of 127.5ms). In the Delay mode, the glass softens, the velocity trip point is reached, and then 300ms of cooling is initiated. The Delay value determines how long the glass is cooled before the hard pull is engaged and continues cooling the glass as the glass is pulled. So by cooling the glass while delaying the hard pull, you can determine how viscous the glass is when it is being pulled. I find this to be a more sophisticated way of controlling the glass when making electrodes. A low Delay value of 40-90 units will expose the glass to less cooling before the hard pull, the glass will be more molten when the hard pull engages and this will result 10