Membrane Test tool in Clampex

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continuously-co...
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Membrane Test tool in Clampex

Hello,
   We're trying to set up a rig for patch clamping. We've got the Axopatch 200B amplifier, and Clampex 10.2. The signals and input/output channels seem to be correctly configured. For example, if we put an electrode in the bath, and run seal test from the amplifier, we get square voltage pulses, and corresponding ~square current responses in the oscilloscope window in Clampex. 
   I would like to use the "membrane test" tool in Clampex to monitor the resistance when forming a seal. We configured the input and output signals in the membrane test configuration window, but then when I try to click "membrane test" in the Tools menu, nothing happens... From the Clampex manual, I believe there's supposed to be a dialog box, with buttons for the stages called "bath", "seal", and "cell"... 
  Any help would be greatly appreciated!!

The FFM
The FFM's picture
My first recommendation for

My first recommendation for when configuring a new rig amplifier and headstage with pClamp would be to do this while having the Patch-1U model cell supplied with the amplifier attached to the CV203BU headstage, rather than using an actual patch electrode dipped in the recording chamber or even patched onto a cell.

This will help you troubleshoot all of the set up as if a pipette was in the bath, in cell attached or in whole cell mode, but using essentially a capacitor and resistor set up for which you are certain of the expected values.

Your membrane test may not be working because you have not correctly telegraphed the amplifier with the digidata and have your telegraphs for gain, lowpass filter frequency and whole cell capacitance neutralization set up appropriately.

here is a useful slide show with a step by step of how to set up the software with the digidata and amplifier correctly

http://www.scribd.com/doc/35252457/Setting-Up-Clampex-for-Data-Acquisition

If this is not the problem post some more info and we can take it from there

continuously-co...
continuously-confused's picture
Thank you for your reply. We

Thank you for your reply. We did actually follow that slide show when configuring the software, except that we're using different IN and OUT channels on the digitizer (since the digitizer is doing double-duty with another amplifier, and those channels were in use). We only set up the telegraph for gain, as they did in the slide show - the other telegraphs aren't plugged in. Do you think that could be the problem?

Regarding the model cell... We did test the amplifier circuits with a borrowed model cell, as instructed in the Axopatch manual, but we had to give it back. (Our amplifier is not so much new, as fallen into disuse, and the model cell is missing.) Do you think this is important enough that we should buy a new one?

Thanks again.

The FFM
The FFM's picture
ALWAYS have a model cell in

ALWAYS have a model cell in the lab.

If anything strange starts happening on your set up, the first thing to do it plug in the model cell to troubleshoot.

I'd get a couple of extra BNC cables and hook up you other telegraphs and configure the inputs in the lab bench in clampex for your signal.

If that doesnt fix it I'll think some more tomorrow - let me knowwhat happens

FYI Axopatch 200B manual can be downloaded here

continuously-co...
continuously-confused's picture
Hi,

Hi,
I wanted to let you know that we solved this problem - reinstalling pClamp and running the "reset to program defaults" tool did the trick. Everything seems to be functioning now. We're currently working on reducing some of the noise, and practicing patching with untransfected HEK cells. 

The FFM
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thanks for the update!

thanks for the update!

Good luck patching!