Low Speed Interphase Extract

Described next are the steps involved in preparing an LSE from eggs that have been harvested and washed as described in the Subheading 3.2.3. Preparation of LSE is an intermediate step in the preparation of HSE (Subheading 3.4.) and NPE (Subheading 3.5.). For HSE, we first prepare LSE from the eggs of 6 frogs; for NPE, we prepare LSE from the eggs of 15 frogs.

1. Transfer the washed eggs to Falcon 2059 tubes. Allow the eggs to settle and aspirate the supernatant. Pack the eggs by gentle centrifugation at 1100 rpm (200g) in a clinical centrifuge for 1 min at room temperature. Aspirate as much supernatant as possible.

2. Add 0.5 ||L protease inhibitor mix and 0.5 ||L 5 mg/mL cytochalasin per milliliter of packed eggs on top of the packed eggs. Do not put the eggs on ice.

3. Crush the eggs by centrifugation for 20 min at 11,000 rpm (20,000g) in a Sorvall HB4 or HB6 rotor at 4°C. Precool the centrifuge to 4°C but keep the rotor and eggs at room temperature until the spin is begun to ensure optimal activation of the eggs. For all subsequent steps, keep the extract on ice.

4. Transfer the tubes to ice. For each tube, puncture the side with a 21-gage needle just above the mitochondrial layer, remove the needle, and insert a fresh needle attached to a 5-mL syringe in the same hole (Fig. 1C). Withdraw as much of the cytoplasm or LSE as possible until just before the pigment granules and lipid begin to enter the syringe. Typically, for a Falcon 2059 tube with 13 mL of packed eggs, we recover 4 to 5 mL of LSE. Combine the LSE fractions from each tube in a 50-mL tube on ice (see Note 4).

5. For each milliliter of LSE, add 5 ||L 10 mg/mL cycloheximide, 1 ||L 1000X protease inhibitor mix, 1 ||L 5 mg/mL cytochalasin, and 1 ||L 1 M DTT. Mix thoroughly by gentle inversion. The yield of LSE is typically 1 to 2 mL per injected frog.

6. Immediately follow the protocol for HSE (see Subheading 3.4.) or NPE (see Subheading 3.5.).

3.4. High-Speed Interphase Extract

This subheading describes how to prepare HSE using freshly prepared LSE (described in Subheading 3.3.). All steps are carried out on ice unless otherwise noted.

1. Transfer the freshly prepared LSE (prepared from the eggs of six frogs) from Subheading 3.3. into four 2.5-mL thin-walled ultracentrifuge tubes (cat. no. 347356; Beckman) and centrifuge for 90 min at 55,000 rpm [260,000g]) in the TLS55 rotor at 2°C. As shown in Fig. 1B, the extract separates into a thin layer of lipids, clarified egg cytosol (the HSE), a membrane fraction, mitochondria, and glycogen.

2. Remove the lipid layer by aspiration with a very thin pipet (i.e., a gel-loading pipet tip).

3. Transfer the HSE from the top of the tube to a new ultracentrifuge tube using a cut-off P1000 pipet tip.

4. If the extract is contaminated with membranes or other debris, recentrifuge as in step 1 for 30 min.

5. Remove the top lipid layer as in step 2, if necessary, and harvest the HSE as in step 3. Freeze the extract as one-use aliquots (50 ||L) by submerging tubes in liquid N2. Store the extract at -70°C, at which it can be stable for up to several years.

3.5. Nucleoplasmic Extract

Subheadings 3.5.1. to 3.5.4. describe how to prepare an NPE extract using freshly prepared LSE described in Subheading 3.3. This includes (1) preparing the nuclear assembly reactions, (2) monitoring nuclear assembly and growth, (3) harvesting the nuclei, and (4) isolation of the nucleoplasm. All steps are carried out on ice unless otherwise noted.

3.5.1. Preparing the Nuclear Assembly Reactions

1. Note the final volume of the freshly prepared LSE (from the eggs of 15 frogs) from Subheading 3.3. and add 0.15 vol of ELB and 1/1500 vol of 5 mg/mL nocodozole. Mix gently yet thoroughly by inversion. Avoid introducing air bubbles.

2. Transfer the extract to new Falcon 2059 tubes and centrifuge for 20 min at 11,000 rpm (20,000g) in a Sorvall HB4 or HB6 rotor at 4°C.

3. Carefully and completely remove the residual lipids and brown material forming the top layer by aspiration with a P200 tip attached to a vacuum line. It is normal to lose about 0.5 mL of extract during this step.

4. Gently decant the extract into a 50-mL conical tube, taking care not to decant any of the dark granular material from the pellet. About 1 mL will have to be left behind per tube to avoid decanting any of the pellet material.

5. Per 1 mL of LSE, add 10 |L 0.2 M ATP, 20 |L 1 M PC, and 1 |L 5 mg/mL creatine kinase. Mix gently yet thoroughly by inversion.

6. Transfer the extract to 5-mL Falcon 2063 tubes and allow to warm to room temperature (22-23°C, about 5 min). Add at least 4 mL but not more than 4.7 mL to each tube.

7. To each tube, add 90 |L 200,000/|L demembranated sperm (Subheading 3.1.). To thoroughly resuspend the sperm in the extract, first add 1 mL of extract from the 2063 tube to

DAPI Stain

Phase Contrast

Q

Fig. 2. Nuclear assembly in LSE. 1-|lL aliquots of a nuclear assembly reaction are taken at the indicated time-points (in minutes), fixed with Hoechst dye, and visualized at 40x magnification on a fluorescence microscope. As the nucleus matures, the DNA staining pattern changes from diffuse to punctate. The mature nucleus shown at the 100-min time-point is 30 |m in diameter.

the tube with 90 |L of sperm. Pipet the mixture 15 times with a P1000 pipet tip and transfer the mixture back to the Falcon 2063 tube. Cap the tube and invert 10 times to distribute the sperm evenly. Repeat for each tube of extract.

3.5.2. Monitoring Nuclear Assembly and Growth

1. Incubate the nuclear assembly reactions at room temperature (22-23°C), gently inverting the tubes approximately once every 10 min.

2. At 20-min intervals, monitor nuclear assembly by mixing 1 | L of each assembly reaction with 1 | L Hoechst fix solution on a microscope slide. Cover each sample with a cover slip and examine the nuclei using a 40x oil immersion objective in the DAPI channel (Fig. 2).

3. Allow nuclei to grow to an average diameter of 25 to 30 | m, which should take about 90 min.

3.5.3. Harvesting the Nuclei

1. Transfer the nuclear assembly reactions from the Falcon 2063 tubes to 13 x 100 mm disposable glass tubes (cat. no. 60825-924; VWR) on ice, combining the contents of two nuclear assembly reactions into each glass tube. Place the glass tubes inside Falcon 2059 tubes containing 3 mL cold water to create a jacket that prevents the glass from cracking. Adjust the level of water in each 2059 tube so that the meniscus is at the same height as the meniscus of the nuclear assembly reactions. Harvest the nuclei by centrifugation for 3 min at 11,000 rpm (20,000g) in a Sorvall HB4 or HB6 rotor at 4°C. A gray, viscous layer of nuclei about 2 to 4 mm thick will form at the top of the glass tubes (Fig. 1D). Transfer the tubes to ice (see Note 5).

2. Remove the entire layer of nuclei to a new 1.5-mL tube on ice, combining the nuclei from all the tubes. The nuclei layer is very viscous and is sometimes difficult to separate from the cytoplasmic layer beneath it. It is most effectively removed by using a P200 Pipetman and a cut-off P200 pipet tip, slowly rotating the tube as the layer is very slowly withdrawn from the perimeter of the tube. When no more nuclei can be harvested this way, continue withdrawing from a smaller radius around the center axis of the tube, working inward. Finally, there may come a point when nuclei remain but are inseparable from the underlying cytoplasm. In this case, transfer the uppermost approx 500 |L of the nuclear assembly reaction (which may appear as a viscous, darker brown fraction) to a 0.5-mL microcentrifuge tube and centrifuge at top speed for 2 min. Frequently, this results in a clear layer of nuclei that is easily harvested. Although the underlying cytoplasm is not inhibitory for DNA replication, contamination of the nuclei with this fraction should be minimized because it will dilute the nucleoplasm recovered from the nuclei.

3.5.4. Isolation of the Nucleoplasm

1. Transfer the pooled nuclei to 5 x 20 mm tubes (cat. no. 342630; Beckman) using a cut-off P200 pipet tip. Centrifuge the nuclei in a Beckman Optima MAX-E ultracentrifuge using a TLS55 swinging bucket rotor (Teflon adaptors [cat. no. 358614; Beckman] are required for the 5 x 20 mm tubes) for 30 min at 55,000 rpm (260,000g) at 2°C.

2. Remove the tubes to ice. Remove any lipids that may be visible on the top of the tubes by careful aspiration with a narrow-tip gel-loading pipet tip attached to a vacuum line. Care must be taken not to remove a significant volume of the clear nucleoplasm. Using a P200 pipet tip, carefully transfer the nucleoplasm to a new tube, avoiding contamination by the pellet of membranes and chromatin at the bottom of the tube.

3. Freeze the NPE in 20-| L aliquots in liquid N2 and store at -70°C. Optimal yields of NPE are 30 to 40 | L per milliliter of nuclear assembly reaction. The NPE is stable for several years at -70°C, but it loses approx 50% activity for every freeze-thaw cycle.

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