I have a narishige PC-10 puller for the job, and haven´t been able to find the right settings in order to obtain a pulled glass pipette with 25-40 um diameter and over a centimeter long in order to reach the hypothalamus in rats. I can get pipettes with a long tip, of about 2 cm long but too narrow (I've also tried breaking their points until I get the right diameter, but by then their points are broken to about 1 cm or less) and also shorter pipettes with 0.9-0.8 cm long points and with the rigth diameter, which are to short for the job.
Does anyone have some settings or tips for working with this specific puller that can help me achieve the pipette I need?
Hi Trinidad Montero ,
I used to do this in mice. So, much smaller brains.
However, I always found the results of the narishige PC-10 to be quite variable. This was probably more to do with the lab's air conditioning system being above the puller than the puller itself. When I switched to a Sutter Flaming Brown puller, I could consistently pull pipettes that were of a sufficient length and diameter for viral injections.
I also used a polisher to grind a 'needle-like' tip to my pipettes to aid in pushing through the brain tissue for better delivery, have you tried this?
Why do you need 25-40 um diameter? Can you get away with using smaller or does the pipette get blocked?
i don't now the diameter size of the final pipette point, but we use borosilicate 10 cm tubes (OD 1.5mm ID 0.86mm) and the program is: [for a Ramp=633]
630(heat)-40(pull) -80(vel)-50(delay)-500(pressure), delay mode clicked and safe heat clicked. only one line, no repetitions. we place the glass with a short end in one side and long in the other side (so we have only one usable pipette). then we fill the pipette with the virus and cut the tip with forceps, little by little until we see a drop going out when we apply pressure with the pico spritzer. otherwise we have also another system that is a hamilton syringe with removable needles, if you want the details let me know. for this version we have a syringe dispenser instead of a pico spritzer.
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