problem with break the membrane

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calciumcurrent
calciumcurrent's picture
problem with break the membrane

I work on rat aortic smooth muscle cell with P2-P6 with patch clamp. The pipette resistance is around 3 MΩ in the solution. I can get a nice seal (above 2GΩ), but when breaking the membrane the leak is too large to record anything. How to decrease the leak? Thanks a lot!

Fraser Moss
Fraser Moss's picture
It sounds like you're just

It sounds like you're just being a little too rough when you break in and go to whole-cell mode.
It takes practice.
If you are using a syringe to give suction , try to use a smaller volume syringe which will give less suctions per unit volume suction applied.
Or try mouth applied suction.  Personally I have always found I have greater control over the acquisition of a seal/going whole cell using mouth suction.
Also look on your amplifier.  See is it has a "zap" function.  This allows you to apply a small and fast voltage jump pulse to the cell as you try to go whole cell.  If you apply a very gentle amount of suction and a small Zap this can help you go whole cell and stay in the GOhm - high hundreds of Mega Ohm range for you seal.
 
Good luck!

calciumcurrent
calciumcurrent's picture
Thank you very much! I use 1

Thank you very much! I use 1 ml syringe to break the membrane. As you said I need more practise and I want to try use the zap.

xiaooyao517
xiaooyao517's picture
Dear  frasermoss

Dear  frasermoss
         I have a question, when breaking  the membrance I find the current become above zero.Can you tell me why it is?

The FFM
The FFM's picture
 xiaooyao517

 xiaooyao517

I'd need to know which cell line you are working with and what is expressed in the cells that you are patching.

By above zero do you mean that you get a positive membrane current?