Nuclear Isolation

After the injections are finished, the next major step is nuclear isolation. This step is imperative to the assay, given the fact that cytoplasmic content would smear the gels, hindering us from analyzing the splicing pattern. Using nuclear isolation followed by RNA extraction, we can obtain a clean nuclear RNA sample that enables us to analyze splicing profile accurately. It should be noted that when doing nuclear isolations, oocytes are healthy, the buffer is pristine, and forceps are sharp and precise (see Note 18). Described next is the protocol for nuclear isolation.

3.4.1. Nuclear Isolation Protocol (Fig. 7A)

1. Transfer the oocytes from 1X MBS buffer to a new Petri dish containing 5:1 isolation buffer.

2. Gently hold the oocytes with a pair of forceps so that the animal hemisphere is on the top.

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