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6. 7. 8. 9. Thermal cycler (Applied Biosystems, Foster City, CA; model 9700). Agarose gel apparatus and reagents. Isogen-LS (100 mL; Wako, Osaka, Japan; cat. no. 311-02621) (see Note 1). Chloroform (500 mL; Sigma, St. Louis, MO; cat. no. 05-3400-5). 2-Propanol (500 mL; Sigma, cat. no. 15-2320-5). Diethyl pyrocarbonate (DEPC)-H2O. Oligotex-dT30 Kit (Roche, Mannheim, Germany; cat. no. 489991). 70 and 100% ethanol. SuperScript First-Strand Synthesis System for reverse transcriptase polymerase chain reaction (RT-PCR) (Invitrogen, Carlsbad, CA; cat.

4. Perform the following steps in a biological safety cabinet. 5. To avoid cell loss, try to keep the drop diameter as small as possible. 6. Healthy blastomeres are recognized because they undergo cell division and show amoebalike movement by forming pseudopodia (Fig. 1C). 7. Appearance of pigment cells at d 3 of culture from some strains/lines seems to be independent of cell density. 8. Subculture by trypsinization, freezing, and thawing of medaka ES cells is done essentially according to standard protocols for animal cell culture.

Remove the bottom cap and allow the sonicate to flow through the column. 7. Wash the matrix by adding 150 mL cold (4°C) 1X PBS. Allow the column to drain. The GST-rchLIF fusion protein will have bound to the glutathione-Sepharose matrix. 8. 8 mL of ice-cold PreScission cleavage buffer. 9. Replace the top cap and gently rotate (10 cycles/min) the suspension at 4°C for 14 h. 10. Remove the bottom cap and collect the eluate in 1-mL fraction in new tubes. 11. Analyze by SDS-PAGE to estimate the yield and purity of every fraction (see Fig.

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Biochemistry - Board Review Series [index at end] by D. Marks

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