We are working on a P-97 puller and we are using it to do whole cell recording in DRG. We have 2 types of pipettes (borosilicate glass ID: 1.1 OD: 1.5 and ID: 0.86 OD: 1.5 ) as it showed in the pic, we find difficulty to set the parameters and sometimes we get different pipette resistance in spite of using the same parameters.
we followed the program suggested in the cookbook (Ramp heat, Pull , velocity and time ). we have tried to modify all parameters but that didn´t help.
if someone working on the same instrument or having any recommendations about the setting parameters I will be thankful.
Hi Ahmed,
it is not only the shape of the pipette tip which is important, also the solutions and relationship between intra- and extracellular solutions. Check that first with a easy to measure cell line - if possible (like HEK293 cells) and then optimize your pipettes.
Good luck ;)
There is a Sutter cook book for that instrument. Hoever hou habe to perform a test ramp, and use the ramo test value for heat (less ten units), and if you want short tips do not use a high strength of pull and reduce heath.. Play with that.
Wolf Berger thank you for your reply
The solutions we are using for external
NaCl 140, KCl 5, D-glucose 10, CaCl2 2.5, MgCl2 1.2, HEPES-ACID 10,
PH 7.4 adjusted with1 M NaoH
for internal solution KCl 130, NaCl 10, HEPES-ACID 10, Mg-ATP 10, EGTA 5,
D-glucose 10, PH 7.3 – 7.4 adjusted with 1 M KOH.
we followed Sutter cookbook parameters but we can't get Gigaseal, we don't know what is the exact problem.
Enrique Soto than you,
i did ramp test and all the recommended parameters in the Sutter cookbook was performed , but i can't form Gigaseal . So I ask if there is standardized parameters to get good shape and resistance of the pipette to work on DRG.
NO standardized parameters. Use them about the recommendations in the cookbook. Which electrode resistance are you searching for that depends on the cell type and cell size you intend to record.
Good morning Ahmed,
thanks for sharing your solutions. I´m now back in the office. I think I found the critical thing..the relationship of intra to external solution is wrong! The osmolarity of your internal solution is ~ 295mOsmol and ~310mOsmol for your external solution. So you will never get a seal. Don´t add glucose to your internal solution and lower your ATP to 5mM this is still enough and finally lower your internal KCl to 125mM so in summary you´ll get a ~290mOsmol solution. With 3-3.5MOhm pipettes it should work.
Happy working ;)
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