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By Paul T. Riegel

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Cells are passaged by manual scraping or collagenase IV treatment. In our experience, Cre protein transduction into hES cells is most efficient if the colonies are very small, ideally clumps of few cells. Therefore, the hES cell colonies are dissociated using accutase II (PAA Laboratories) prior transduction. Accutase II has the advantage that it selectively detaches the hES cells from feeder cells if incubation does not exceed 30 min. The single-cell suspension is plated on MEFs again and allowed to adhere for 24 h.

4. 5. 6. 7. 8. 50% Ni-NTA agarose (Qiagen). Econo-Pac columns (Bio-Rad). Benzonase (Novagen). Lysozyme solution: dissolve lysozyme (Sigma) in ×1 buffer A to a concentration of 80 mg/ml. Always prepare freshly before use. 0. Sterilize by filtration and store at room temperature. TT buffer: 2 M disodium tartrate, ×1 buffer A, 15 mM imidazole. Sterilize by filtration and store at 4 C. Washing buffer: 500 mM NaCl, ×1 buffer A, 15 mM imidazole. Store at 4 C. Elution buffer: 500 mM NaCl, ×1 buffer A, 250 mM imidazole.

Protein Expression 1. LB medium: 10 g tryptone from caseine, tryptic digest (Roth), 5 g yeast extract (Roth), 5 g NaCl. Suspend in 1 l double-distilled water and autoclave at 121 C for 20 min. Store at 4 C in the dark. 20 L. Nolden et al. 2. TB medium for protein expression: 12 g tryptone from caseine, tryptic digest (Roth), 24 g yeast extract (Roth), 4 ml glycerol (100%), 2 31 g KH2 PO4 12 54 g K 2 HPO4 . Suspend in 1 l double-distilled water and autoclave at 121 C for 20 min. Store at 4 C in the dark.

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Border Watch (Greyhawk Adventures Game Module) by Paul T. Riegel


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