This forum is great, I recently found it and thoroughly enjoying it. So I decided to ask something myself, as I have some problems with patching.
I´m patching a HEK cell line stably expressing TRPM8. Forming a seal is not a problem whatsoever, especially when using fire-polished pipets I get seals real fast and with a high resistance (4 GOhm or more, is that normal?) With un-polished pipets this is lower, around 2 MOhm and seals form less easily.
The real problem is after I get the seal. It is quite impossible to go to whole cell with the fire-polished pipets. With the non-polished ones it is a bit easier, but still hard. I open the cell by applying suction with the mouth. Any suggestion as to what might be going wrong here? And to make it a bit harder still, I recently switched from regular trypsin to a different type of trypsin, which leaves the cells looking very nice and making forming a seal easier.
Basically the problem is that sealing got a a lot easier, but opening a lot harder.
Any suggestions are welcome and I´m sorry for the slightly incoherent story, but it´s been a long day ;)
Edit: some aditional information
The problem is not that it is impossible to open the seal, When even the slightest zap is applied I lose the seal. With gentle suction nothing happens until I just ¨kiss¨ it a little bit harder and voila, I lose the seal.
pipet resistance is generally between 3 and 4 MOhm
bath temperature is 32 degrees Celcius