Breaking / polishing injection micropipettes

[NOTE]: Pipettes that come out of the puller are not ready to be used in an experiment or for RNA injection. There is no hole in the newly formed tip, and so they need to be "broke" in order to be usable. This page walks you through both breaking and polishing.

Breaking

Remove the two freshly pulled pipettes from the P-97 puller.
If it's an RNA injection pipette, place one of the fresh pipettes into the "unbroken pipette" dishes for now. If it's a TEVC pipette, place it somewhere safe so that the tip will not break.
Lay a Kimwipe flat out on the table. This is what we'll use for the "breaking."
Very lightly, touch the pointy tip of the pipette to the wipe and softly drag it a few millimeters along. [The action is similar to making a graphite dash with a pencil on paper.]
The pipette should now be broken, but it's far too small to see with the naked eye. We'll need to check it with a microscope.
Turn on the Micro Forge MF-830 (it's to the left of the RNA injection microscope).
Take the now broken pipette and clip it into the MF-830 Micro Forge stand There are two silver clips, but the pipette will only clip into the bottom one while laying over the top one. There will be an audible click when doing this, and this lets you know that the pipette is now secure. See image below.

B is pointing where the clip that the pipette will lay on top of (not clipped into).

A is pointing at the clip that the pipette will clip into.

The line between them is where the pipette should be when in place.

8. The small heating ring for the rig should already be in place, so avoid moving it towards or backwards with from you unless you've had experience. There should be a sticky note over the knob that does this.

9. Using the knobs that control the pipette holder, move the pipette downwards until it enters your field of vision in the microscope. You may need to move it left or right as well.

10. Adjust pipette until it lays within your field of vision and is flush along the unit measurement line that should also be in your field of vision. We're going to count the number of units that the broken edge of the pipette takes up.

11. Ideally, injection pipettes should be within the 2 to5 units' range for diameter. There are benefits and drawbacks to both sides of this range.

12. If the broken pipette falls into this range, then the "breaking" was successful and you can move onto polishing. If the tip is smaller than two units, then you can break it further by repeating Step #4. Check the break again after this. If the tip is much larger than five units, discard it into the "Used pipettes" waste container.

Polishing

"Polishing" refers to heating the tip of a micropipette with the metal ring from the Micro Forge MF-830. We do this in order to clear away Kimwipe or glass debris and to sanitize the tip of the micropipette. [NOTE: It's not necessarily mandatory, but it is a quality insurance step, and may help you get better data than you would without this step, as it limits the variables that are incontrollable.]_____________________________________________________________________________________________________________________________

Select a broken, pre-polished pipette and turn on the MF-830 Micro Forge. Identify the Forge's pedal on the floor and move it close to your feet.
Clip in the pipette into the MF-830 just like how you would if you were checking for a pipette tip break.
While looking through the lenses, adjust the knobs that control the pipette support so that the pipette moves downward. You should only need the 5M objective lens for this (orange stripe).
Switch back and forth from looking through the eyepiece into frame to looking normally. This is so you don't end up crashing the pipette into the ring from lack of depth perception. The ring is fragile so be sure to not let it touch the pipette.
Stop moving the pipette whenever you can see the tip come into frame. You will want a large distance between the ring and the pipette for this next part.
Keeping your eyes on the pipette and ring, step on the green pedal below the table. This activates an electrical current through the metal ring, which heats it up and causes it to expand.
See how much space in the frame that the ring takes up while being heated, and keep in mind how far it needs to be from the ring.
Step off the pedal to halt the current.
Move the pipette downward until it's about 5 units above the edge of the ring.
Switch the objective lens to 35M (blue stripe). You should still be able to see the tip of the pipette, but if you can't, adjust the focuses and knobs in such a way so that you can.
Remembering how far the ring expands to when heated, move the pipette so that it's a safe distance from the edge of the ring. It's safer to be too far than it is too close. You can always move it closer
Once you're ready, step on the pedal again and watch the pipette. You should see the rough edges of the break melt into a uniform one, and any debris that was visible should melt away.
It's now polished. Carefully remove it from the Micro Forge stand and rehome it into the "polished and broken" pipette dish.
Reset the focuses and turn off the Micro Forge.
Re-cover the Micro Forge.

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