膜片吸液管和夏普电极与一个可编程的拖轮

Published 10/08/2008
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Biology

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Summary

该视频演示了如何使用一个可编程的车夫电补丁的移液器和锋利的电极。可以使用同样的程序,作出各种玻璃工具,包括注射针头。

Cite this Article

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L. Brown, A., E. Johnson, B., B. Goodman, M. Making Patch-pipettes and Sharp Electrodes with a Programmable Puller. J. Vis. Exp. (20), e939, doi:10.3791/939 (2008).

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Abstract

玻璃微电极(也称为移液器)已经几十年了电的主力。如今,这样的吸液管是由玻璃毛细管使用一个可编程的车夫。这种仪器的热量使用金属丝或激光,并绘制出重力,马达或两者同时使用玻璃毛细管。膜片钳记录滴管使用唯一的热量和重力形成的,而锋利的电极细胞内记录,用热,重力和电机的组合。用来使细胞内记录移液器的过程是类似的用于各种应用,包括的cRNA注射到非洲爪蟾卵母细胞的注射针头。一般情况下,玻璃毛细管<直径1.2毫米,是用来做膜片钳记录的移液器,而窄的玻璃是用于细胞内记录(外径为1.0毫米)。对于每一个工具,拉马是编程略有不同。该视频演示如何使两种录音移液器使用预先确定的车夫方案。

Protocol

拉移液器

电极使用拉马等,作为萨特的P - 97火焰山/布朗,拉约10-20移液器。

  1. 选择你的毛细血管玻璃。我们使用高硼硅毛细管(萨特BF150 - 110 - 10,1.5毫米外径,内径1.1毫米,长10cm)。商店的玻璃,仔细,所以保持清洁和无尘。
  2. 设计一个拉动的程序。我们采用5步程序,在每一步,降热和速度和小拉的最后一步。 Sutter的吸管的食谱是一个合适的方案开发很好的参考。
  3. 检查在显微镜下观察,以确定开口直径和平滑的枪头。舍弃粗糙,凹凸不平,或不规则的小窍门。一个很好的拉动协议应确保这些都是罕见的。对于标准的膜片钳记录,提示开口应在直径1-3微米。一个陡峭的锥形先端钝,导致较低的电阻相同的开口直径。形状和大小可以修改的压力抛光。

消防抛光移液器

  1. 成立由脚踏板控制的加热丝铂金抛光钻机(microforge)。一个有用的工具包是ALA的科学(CPM - 2)或类似的设备可组装头痛医头,脚痛医脚。
  2. 可选:大衣与绝缘体,以减少电容和改善噪声特性吸管。我们使用牙科用蜡。 Sylgard 184™(聚二甲基硅氧烷弹性体,又称作为硅橡胶)也是一种选择。
  3. 如果用蜡,使附近的一个小的熔融供应。进入移液器移液器保持蜡背面的空气压力,提示简要浸入液体蜡和删除。随着Sylgard,商店准备的弹性体在冷冻分装油漆的枪头,在显微镜下。热固化的弹性体。
  4. 将吸管抛光设备(涂层或不)〜50微米的灯丝带来的小费。请记住,灯丝加热时,将扩大。
  5. 建议 :按照单独的协议为“压力抛光”改变形状以获得最佳的性能和尖端直径吸管。
  6. 一个简短的热脉冲(1-2秒),是足以消除吸管尖蜡和光滑的玻璃。
  7. 成品吸管放置在一个封闭的盒子,以防止灰尘,重复成功移液器10。

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Discussion

协议说明这里是在电生理实验室的日常使用,也可用于细胞和动物的注射针头。一个可编程的车夫,很容易使多种用途的移液器。重视和关怀,你的车夫长丝将持续一年或一年以上。祝您的实验。

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Acknowledgements

我们感谢以下资助机构的支持和基础:美国国立卫生研究院,唐纳德B.肌肉萎缩症协会,美国国家科学基金会,美国心脏协会,和迪莉娅E.巴克斯特基金会,Klingenstein基金和神经科学的麦克奈特基金会。

Materials

Name Type Company Catalog Number Comments
Micropipette Puller Instrument Sutter Instrument Co. P-97 Or similar instrument (e.g. Sutter P-87 or P-2000)
Glass Capillaries Reagent Sutter Instrument Co. BF150-86-10 Or, similar capillary glass. To make filling the pipette easier, use a capillary with a glass filament.

DOWNLOAD MATERIALS LIST

References

  1. Sutter Instrument, P-97 Pipette Cookbook. Sutter Instrument. http://www.sutter.com/contact/faqs/pipette_cookbook.pdf (2008).

Comments

6 Comments

  1. 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.  

    Reply
    Posted by: Anonymous
    October 31, 2008 - 1:19 AM
  2. 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

    Reply
    Posted by: Anonymous
    October 31, 2008 - 2:36 PM
  3. 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.

    Reply
    Posted by: Haluk B.
    February 6, 2009 - 4:20 PM
  4. 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.

    Reply
    Posted by: Anonymous
    February 6, 2009 - 5:54 PM
  5. Thank you very much Mr. B.Johnson. Sincerely....

    Reply
    Posted by: Haluk B.
    February 7, 2009 - 9:53 PM
  6. Dear authors,
    Thank you for this great video. May I ask which dental wax you are using? Also, what is the temperature of the wax? Your help is greatly appreciated.

    Reply
    Posted by: Taro K.
    September 21, 2014 - 9:03 AM

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