We use sutter P-1000 Pipette Puller with 1.00mm -0.50mm filament for C.elegans microinjection.
But the taper of needle tends to bend when injecting worm :(. Could you recommend better parameter settings we can follow?
what is your heat setting? try turning it down 5-10%
the heat setting we used is 740, could you provide us with the detailed setting working well in your lab? thanks a lot
something like that should be good. the heat setting should be not too far away from the value you get when you do a ramp check on your pipettes
we get satisfying needle according to your setting, thanks so much for your kindness ;D
you should first make a ramp test because sometimes the filament tends to alter along the time! then you can try to down a little the heat temperature,
and vary the pull so that your taper is not so long that bends!
My settings are:
ramp test: 499
Pull: 50 (60-80)
Time: 135 (130-170)
hope it works!
We recently ran into some needle puller problems with the Sutter P-97. This thread is a useful guide to the art of needle pulling, so I thought it was worth resuscitating and adding our experiences.
Capillaries: World Precision Instruments, Item #: 1B100F-4
I strongly recommend getting the user’s manual here. As with asking for directions when you are lost, manuals are generally a good thing. This one certainly is. And, unusually, it is well written and helpful.
As noted by lorkitas above, running a ramp test in really important. This will tell you how well your heating filament is working. I suspect ours is old (we’ve been using it for the 5 years I have been here). Not surprisingly, the ramp test gave us a value of 635. That’s substantially higher than noted by users above. Again, we suspect that is because the filament is old.
Also very useful is the “Parameter Adjustment” section on page 29. We found this to be spot on. (NOTE: it is helpful if you know the shape of an effective needle. Have one of your own, get one from a neighbor, or have somebody send you a picture of one!)
The impact of “Heat,” “Strength,” “Velocity,” and “Time” parameters are well defined for injection vs. micropipettes. Isn’t that great? We found that the ranges provided in the manual were consistent with getting us an effective needle. Hooray! Some trial and error still needed.
Heat: "The HEAT setting will affect the length and tip size of the pipette. Generally speaking, higher HEAT settings tend to give longer and finer tips. For trough filaments, the recommended starting HEAT value is the ramp test value plus 15 units. For box filaments the recommended starting HEAT value is the ramp test value. (We think these are trough filaments, but that is not clearly defined. Sorry.) The program listed above will typically have heat on for 5 to 8 seconds after the < PULL > key is pressed. If the time is longer than eight seconds, and you are trying to pull a fine micropipette, increase the HEAT in 5 unit increments until the pull takes place in less than eight seconds. If the pull occurs in less than three seconds, decrease the HEAT until the pull takes place in 4 to 8 seconds. For the best micropipette reproducibility with the finest tips, you should select a HEAT value that melts the glass in 5 to 6 seconds. For microinjection needles you should select a HEAT value that melts the glass in about 7 seconds or longer.
Strength: "Low values of PULL strength in the range of 40-75 will give larger tips appropriate for injection needles, while settings between 120-250 give smaller tips appropriate for micropipettes.
Velocity: "The VELOCITY value determines the point at which the heat is turned off. VELOCITYreflects the speed at which the two carrier bars are moving during the weak pull. The lower the VELOCITY value the slower the speed of the bars when the trip point occurs. Although VELOCITY can safely be set to any value from 1-255, all values over a maximal trip point
(about 150) will produce equivalent results. As the pull progresses, the speed of the carrier bars … reaches a point where further increases in the VELOCITY trip point will not change the time point at which the heat is turned off. VELOCITY is typically set between 80 to 120 for micropipettes or 50-80 for microinjection needles.
Time: "The TIME parameter controls the length of time the cooling air is active. … increasing cooling TIME values beyond a certain range (typically between 150 to 200) will have no effect. Values of TIME shorter than this same range will cool the glass less as the tip is being formed and lead to a longer taper. However, once TIME values become too short (values in the range 110 to 130) cooling becomes ineffective. The glass will not form a tip and instead forms a wispy fiber.