Hi. I am having issue with maintaining positive pressure. This was not the case earlier but suddenly it has become the major factor in deciding if the recording will be a success or a failure. Basically, I am getting a pressure kickback where the positive pressure is lost in few seconds before I am able to release it for seal to happen and this loss in pressure kicks back creating a negative pressure and sucking things or cell debris into the pipette. When this happens the pipette gets clogged to a point that any further suction of any amount won't work and the pipette cannot be used.
I have changed the tubing, the stop cock, cleaned the pipette holder and everything else I can do but it does not seem to solve the issue completely. The problem eventually comes back. I am unable to pin point the source of the problem but I can think of few culprits:
1. Pipette is too narrow, which I doubt is the problem here ( ppt resistance of 3-5 megaohms)
2. Pipette sol is the problem- I did suspect it but have made new solutions often but it tends to come back. The first few days after a fresh solution seems to work better but the problem comes back. I wonder if crystallization is happening while the ppt solution is at -20 degrees. I have also tried sonicating the solution with few quick bursts
3. Positive pressure is too strong.
My feeling is that it is more than the factors above. If anyone has or have had similar experience and were able to solve the issue then please let me know.
Thanks
Jimmy
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