Tornando-Patch pipetas e Eletrodos Sharp com um extrator programável

This video is just misleading any new students that pulling patch pipetts are that simple. In reality, without knowing how to program the puller, how to choose right filaments-box or other types, how to instal and allign the filament..............pipette pulling is really incomplete story.

To all students who will watch this video, I encourage to start with cook book and choose right cappilary tubes for your application. Don't take the words for granted that 1-3 micron tip is good for patching. Also, you don't have to always fire polish the capillary. Readymade fire polish capillary are available as well.

Dear Austin L. Brown, Brandon E. Johnson, Miriam B. Goodman, Department of Molecular and Cellular Physiology, Stanford University

Thanks for demonstrating the art of pulling and making patch type pipettes.

Five things to note:

1) When patching cultured cells, it is best to use BF150-86-10 as you demonstrate in the video. You can use a one line program that loops 4-5 times and it is best to use a midpoint velocity (instructions found on page 26 in the cookbook) to have a stable and reliable program. If for some reason the tip size or resistance is not exactly as you need, you can then write the program out into a 4 or 5 line program and adjust the heat or velocity on the last line to better control the tip morphology. I personally would not create a program with gradually descending heats and velocities as this seems a bit labor intensive and maybe confusing. It could also introduce some variability. But, if it works, stick with it. There are many roads leading to the same point (so to speak), and as long as your program is stable, you will have a high yield of usable pipettes.

2) When pulling patch pipettes for slice or whole tissue, it is best to use thin walled glass BF150-110-10 and a one line program that loops two times instead of 4-5. This will provide a slightly longer  taper and a more gradual approach to the tip. This more gradual taper is best for inserting the pipette through multiple cell/tissue layers as it reduces damage to the tissue.

3) Always run a ramp test when writing a new program or using a program/puller you are not familiar with. When using a box filament (which is best to generate short tapers and high cone angles), it is best to use the ramp value for the heat setting. When using a trough filament (which generates longer tapers) it is best to use ramp+10 or ramp+15 for your heat setting.

4) The NEW P-1000 pipette puller (which is being shown at the Neuroscience meeting in December 2008 in DC) has a Cookbook feature on the touch screen menu where you can search for and install a cookbook program. Starting programs can be found by designating the filament type, glass size, and application. It will then produce a program with which to get started....so there is less guess work! It also has a "Safe Heat Mode" which will reduce the incidence of burning out the filament.

5) When pulling sharp pipettes, you can go to the General Look Up Tables at the end of the Cookbook. Here you will find Type, A, B, C, D, and E programs. I recommend starting with a Type A for PAtch, and a Type B or C for sharp electrodes where the resistances are >20 Megohms.

If anyone out there need more help, feel free to call Sutter 415-883-0128 and ask for me (Adair) and I will be happy to help with any additional concerns.

Sincerely, Adair

To examine the obtained pipette (5-10micrometer radii) under a microscope, is the microscope special properties (stereo, invert, ....)? Could you infrom me about it, please? Thank you.

We use a Leica DM IL inverted microscope with a pl fluotar 100x objective.  The key is to have a high magnification, long-working distance objective.

Thank you very much Mr. B.Johnson.

Sincerely....