(old) Injecting Protocol

Injecting Protocol

This is meant to be more of a refresher for the in-person training for injection. Currently we are injecting 0.02% L102C and 50ppm R104C, these should express by day 3 (if the day of injection is day 0).

*Do not forget to wear gloves and eliminase, and wipe down the desk and microscope with alcohol*

Bring the yellow cryo-safe in freezer A down to the -80 freezer. The RNA is in the box with the blue sticky note on the top. When you get back to lab keep it in the freezer.
Look at the injection micropipette, make sure the tip is not broken and there are no air bubbles. Make sure some oil comes out when pushed out. Replace if needed.
Bring MBSH, the injection plate, MBSH+AB+AF, the twelve well plate, a small beaker, and the bottle of filter di water to the tissue culture hood. Pour MBSH in to the injection plate and MBSH+AB+AF in to how ever many wells in the twelve well plate you will be using. Pour filtered di water in to the small beaker, this is what you will use to rinse the plastic pipet with. (We do this since we have had a problem with infection recently, we want the bottles of solution to stay as sterile as possible).
Get the twelve well plate of uninjected oocytes from the incubator. Use the microscope to pick out 10 of the best-looking oocytes and put them in to the injection plate. (In the beginning 1µL is enough to inject ~10 oocytes but with more practice you may be able to inject more.)
Pipet 1µL of RNA on to the glass slide covered in parafilm.
Put the tip of the micropipette in the RNA bubble, push a little oil out, then take up the RNA.
Mark the slide on the parafilm where the RNA was.
Inject (remember: you should see the membrane give way to the pipet and the oil-RNA interface moving when you push the RNA out). Seeing a little “yolk” come out is good.
Put them in the new twelve well plate and write the RNA, your initials, and the date.
Put the RNA in the cryo-safe back in to the -80 freezer.

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