tap\u57f9\u517b\u57fa\u7528\u4e8e\u83b1\u8335\u8863\u85fb\u7684\u57f9\u517b\uff0c\u4ee5\u4e0b\u4e3a\u539f\u7248\u914d\u65b9<\/p>\n
from Gorman, D.S., and R.P. Levine (1965) Proc. Natl. Acad. Sci. USA 54, 1665-1669.
\nThis is probably the most widely-used medium at present for experimental work.<\/p>\n
Make the following stock solutions:
\n1. TAP salts<\/p>\n
NH4Cl\u00a0 \u00a0 \u00a0 \u00a0\u00a0\u00a0\u00a0 \u00a0 \u00a0 \u00a0 15.0 g
\nMgSO4 . 7H2O\u00a0 \u00a0 \u00a0 \u00a0\u00a0\u00a0\u00a0 \u00a0 \u00a0 \u00a0 4.0 g
\nCaCl2 . 2H2O\u00a0 \u00a0 \u00a0 \u00a0\u00a0\u00a0\u00a0 \u00a0 \u00a0 \u00a0 2.0 g
\nwater to 1 liter<\/p>\n
2. phosphate solution<\/p>\n
K2HPO4\u00a0 \u00a0 \u00a0 \u00a0\u00a0\u00a0\u00a0 \u00a0 \u00a0 \u00a0 28.8 g
\nKH2PO4\u00a0 \u00a0 \u00a0 \u00a0\u00a0\u00a0\u00a0 \u00a0 \u00a0 \u00a0 14.4 g
\nwater to 100 ml<\/p>\n
3. Hutner’s trace elements<\/p>\n
To make the final medium, mix the following:
\n2.42 g Tris
\n25 ml solution #1 (salts)
\n0.375 ml solution #2 (phosphate)
\n1.0 ml solution #3 (trace elements)
\n1.0 ml glacial acetic acid
\nwater to 1 liter
\nFor solid medium, add 15 g agar per liter
\nAutoclave.
\nFor Tris-minimal medium omit the acetic acid and titrate the final solution to pH 7.0 with HCl<\/p>\n
Hutner’s trace elements
\nHutner et al. (1950) Proc. Am. Philos. Soc. 94, 152-170
\nThis mixture is used both in TAP and in the Sueoka high salt medium.<\/p>\n
For a detailed analysis of how well this trace elements solution meets the nutritional requirements of C. reinhardtii, see Merchant et al. (2006) Biochim. Biophys. Acta 1763, 578-594.<\/p>\n
For 1 liter final mix, dissolve each compound in the volume of water indicated.
\nThe EDTA should be dissolved in boiling water, and the FeSO4 should be prepared last to avoid oxidation.
\ncompound\u00a0 \u00a0 \u00a0 \u00a0 amount\u00a0 \u00a0 \u00a0 \u00a0 water
\nEDTA disodium salt\u00a0 \u00a0 \u00a0 \u00a0 50 g\u00a0 \u00a0 \u00a0 \u00a0 250 ml
\nZnSO4 . 7 H2O\u00a0 \u00a0 \u00a0 \u00a0 22 g\u00a0 \u00a0 \u00a0 \u00a0 100 ml
\nH3BO3\u00a0 \u00a0 \u00a0 \u00a0 11.4 g\u00a0 \u00a0 \u00a0 \u00a0 200 ml
\nMnCl2 . 4 H2O\u00a0 \u00a0 \u00a0 \u00a0 5.06 g\u00a0 \u00a0 \u00a0 \u00a0 50 ml
\nCoCl2. 6 H2O\u00a0 \u00a0 \u00a0 \u00a0 1.61 g\u00a0 \u00a0 \u00a0 \u00a0 50 ml
\nCuSO4 . 5 H2O\u00a0 \u00a0 \u00a0 \u00a0 1.57 g\u00a0 \u00a0 \u00a0 \u00a0 50 ml
\n(NH4)6Mo7O24. 4 H2O\u00a0 \u00a0 \u00a0 \u00a0 1.10 g\u00a0 \u00a0 \u00a0 \u00a0 50 ml
\nFeSO4. 7 H2O\u00a0 \u00a0 \u00a0 \u00a0 4.99 g\u00a0 \u00a0 \u00a0 \u00a0 50 ml<\/p>\n
Mix all solutions except EDTA. Bring to boil, then add EDTA solution. The mixture should turn green. When everything is dissolved, cool to 70 degrees C. Keeping temperature at 70, add 85 ml hot 20% KOH solution (20 grams \/ 100 ml final volume). Do NOT use NaOH to adjust the pH.<\/p>\n
Bring the final solution to 1 liter total volume. It should be clear green initially. Stopper the flask with a cotton plug and let it stand for 1-2 weeks, shaking it once a day. The solution should eventually turn purple and leave a rust-brown precipitate, which can be removed by filtering through two layers of Whatman#1 filter paper, repeating the filtration if necessary until the solution is clear. Store refrigerated or frozen convenient aliquots. Some people shorten the time for formation of the precipiate by bubbling the solution with filtered air.
\nIf no precipitate forms, the solution is still usable. However, you might want to check the pH in this case and adjust it to around 7.0 using either KOH or HCl as needed.<\/p>\n