Anjul, what you are seeing is far from uncommon. Your suspicion is correct: the mechanical clamping of the pipette in the electrode holder is insufficient. Many electrode holders tend to leave a bit of wiggle room when a pipette is inserted and the front screwed tight. The problem is that the tightening only fixes the pipette at a single point. 

One simple thing to do is to see make sure that the pipette is far enough back in the electrode holder. Many holders have a narrow section (or even a second rubber o-ring) at the back that helps support the pipette. 

A more expensive fix is to buy specially designed pipette holders that clamp the pipette at two or even three points. G23 Instruments are the only company I know of that does this (I have no commercial interest). I know of several labs using their holders for very precise work (patching multiple sites on dendrites) and all users I have spoken to are happy with them:

http://g23instruments.com/

Good luck with your experiments!

 Dear Anjul,

Sounds like you are pretty much in control. There are couple of things  that I can suggest that you may find helpful in solving your problem. First of all make sure that your electrode holder is in good condition, and all the screws are tightening well and easily. Secondly pay attention to the tubing (mouth piece) that you use for pressure application, tubing should be soft and also a bit loose-not too tight to dampen the drift due to pressure. You should not need to apply too much pressure to seal to the membrane, if the membrane is cleaned/debris blown away properly. 1.5 mm ID rubber should be fine, in my case it is not a ring but rather a long (~4-5 mm) rubber sealer. Once you push the electrode all the way into the holder through this rubber seal, nd tighten the screws, the electrode should not move much. Remember that there will always be some drift of the pipette tip with the pressure, as long as its tiny (1-2 micron) and manageable (does not cause significant drop in seal/break in rates).

Best wishes,

Refik

Dear Anjul

There is slight x-y movement when you break the membrane. regarding movement during application of positive pressure, please check your airtable and air pressure. Also try to clean the tube connecting head stage (pipette holder) and syringe. Make sure that the pipette holder is clean and connections are tight.

Good Luck 

Rakesh

Dear Christian, Thanks a lot! I will look into the pipette holders. I am using the manipulators from Scientifica only (patch star), which are very good :)

Dear Refik, Thanks a lot! I did screw the holder tightly. I normally apply negative pressure through a 2 mL plastic syringe instead of using mouth. My seals always break whenever I apply suction through mouth.

Dear Rakesh, Thanks a lot! i will check the tubing and will clean it or else will change it altogether.

Less pressure = less tip movement. If your lab has a manometer, you could measure the actual pressure that you are applying - quite likely this will help you to reduce the pressure (positive on approach and negative on sealing) below the levels you are using now, and to obtain more consistent values across experiments, Reducing pressure has the benefit of disturbing the tissue less.

Dear Anjul,

Alternatively you can use voltage pulses once you form giga-seal to break the membrane (without any suction). See my following publication on that. You can apply this protocol with or without Neurobiotin. In my experience, I find that the recordings are a lot more stable and less likely to re-seal as you are not sucking the cytoplasmic content/organelles/membranes into the pipette tip.

best wishes, Refik

Article Semi-loose seal Neurobiotin electroporation for combined str...

 Dear Nejamin, Thanks a lot. Yes, reducing the pressure actually helps. 

Dear Refik, Thanks a lot! I will see to it. Previously, I tried applying voltage pulses of several mVs for 1 ms to few ms, and also zap (1.5 V) for 50 us to 500 us but it did not help. 

I recommend incrementing voltage pulses of 0.5 to 1 second. duration at 50 to 200 mV range, depending on membrane breakdown of your cells. For details see the paper I have listed above. Zap is often too much and easy to kill or loose the seal.

cheers, Refik

Technical Report Research Methods and Technical Considerations: Answers to ov...