Can anyone help out with settings for the Narishige PC-10 pipette puller that give good needles for microinjection? I have been using a single-pull with the heater set to 55 and all four weights. This setting is the coolest that will actually result in melting of the capillary into two needles. I am using the standard World Precision Instruments 1B100F-4 capillaries and have also tried the Narishige GD-1 needles that came with the puller.
The needles I get are usually open, but with a flow so low that I need to break off the end against a piece of pulled Pastuer pipet. I have found it difficult to get needles that are open enough to get a good flow without breaking it off to the point where too much damage is done to the worm. I also have a lot of problems with needles getting clogged by “worm guts” even though I try to keep the needle flowing while I withdraw it.
I also have access to a Sutter Flaming/Brown P-97 puller in a nearby lab if that would help.
Thanks
-Tim
Hi Tim,
We use the PC-10 capillary puller (step 1 mode, 59 C) with the Narishige GD-1 capillaries. We center the capillaries to get two needles out of each. The needles are closed when pulled, and we open them using a broken coverslip on the injection pad or by dragging the needle across the injection pad. This works most of the time, but it takes some practice.
Best of luck!
Ismar
I also use World Precision Instruments 1B100F-4 capillaries. I pull them on a Sutter Flaming/Brown P-97 puller with these settings: H=925, P=0, V=40, T=250. Not quite ideal, but quite usable needles result (I’d like them a tad longer). I can use both needles from a pull, but they both are closed after pulling. I use a pulled glass capillary to break the needles on, placed at an angle to the needle to bevel the tip. This glass filament can be used repeatedly and provides a smooth, uniform surface on which to break a needle. I overlay the glass filament with oil 1) to be able to see when the needle is getting close to the rod (when the needle breaks the surface of the oil, I switch from low to higher mag- I like to break the needle at the same mag as I inject) and 2) to observe the flow rate from the needle.
In addition to beveling the needle, slipping the needle into the worm at an oblique angle (say 10 vs. 90 degrees) may help reduce damage to the worm. It helps with puncturing the gonad, I think.
I also filter my injection solution, so I don’t get clogging from that source. I don’t keep the needle flowing as I withdraw it (though I check after withdrawing to confirm it’s not clogged. If so, doubling the pressure usually blows out the occlusion).
I’ve made a movie of a needle right after breaking that shows what I think is a good tip and flow rate (also the angle of the glass rod). I’ll send it to you if you’d like (contact me by e-mail. It’s too big to attach here.)
Tina L. Gumienny
Texas A&M HSC
College Station, TX