Notes

1. Culture conditions: Flask volume should be at least four times larger than the culture volume The shaker speed should not be more than 200 rpm Too much shaking increases cell density but it has a negative effect on polymer yield

2. SDS-gel electrophoresis Load the protein samples immediately after boiling Do not store samples on ice, since SDS would precipitate

3 Transmission electron microscopy If unbound osmium tetroxide is not washed properly in subsequent buffer washes, Os will deposit all over the specimen causing Os pepper on sections Proper fixation of tissues is essential for good quality sections on microtome. Hence, follow fixation steps carefully, especially the duration of fixation. Super glue instantly bonds skin; therefore, wear protective gloves

4. Staining for TEM: Uranyl acetate is photosensitive. Try to avoid exposure to light as much as possible and store in the dark at 4°C. Keep the Petri dish covered with aluminum foil during 40 min staining period. Lead citrate solution should be prepared on freshly boiled, double-distilled water since it is extremely sensitive to C02. Use a narrow-necked bottle to avoid exposure to C02 in air and store at 4°C. Exposure to C02 in air results in formation of lead carbonate resulting in deposition of lead carbonate on the sections While staining, do not let grids float on lead citrate. Immerse grids completely inside the stain drop, to avoid exposure to C02 in air You should also hold your breath while handling the grids to prevent C02 exposure to grids NaOH is highly corrosive Do not use near instruments that do not have any protective cover on them.

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