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金虫 (小有名气)

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专业: 环境工程

[求助] TAP培养基配制问题已有1人参与

有没有配过TAP培养基的，我按照如下的配方配制之后，第一次养莱茵衣藻，接种的量比较少，没有长起来。第二次又配制了一次，测了一下PH是4点多。我的疑问是，Tris不是可以缓冲的么，如果不可以缓冲，那肯定是要用氢氧化钠或钾调节PH的，那何不直接加乙酸钠呢？
TAP培养基配方：
Make the following stock solutions:
1. TAP salts                               2. phosphate solution
NH4Cl      15.0 g
MgSO4 . 7H2O      4.0 g
CaCl2 . 2H2O      2.0 g
water to 1 liter
K2HPO4      28.8 g
KH2PO4      14.4 g
water to 100 ml
  3. Hutner's trace elements
For 1 liter final mix, dissolve each compound in the volume of water indicated.
The EDTA should be dissolved in boiling water, and the FeSO4 should be prepared last to avoid oxidation.
compound amount water
EDTA disodium salt 50 g 250 ml
ZnSO4 . 7 H2O 22 g 100 ml
H3BO3 11.4 g 200 ml
MnCl2 . 4 H2O 5.06 g 50 ml
CoCl2. 6 H2O 1.61 g 50 ml
CuSO4 . 5 H2O 1.57 g 50 ml
(NH4)6Mo7O24. 4 H2O 1.10 g 50 ml
FeSO4. 7 H2O 4.99 g 50 ml
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.
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.
If 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.
To prepare sulfur-free trace elements for hydrogen generation, the sulfate salts can be replaced with equimolar chloride salts (ZnCl2 10.0 g; CuCl2 . 2 H2O 1.00 g; FeCl2 . 4 H2O, 3.60 g). .
To make the final medium, mix the following:
2.42 g Tris
25 ml solution #1 (salts)
0.375 ml solution #2 (phosphate)
1.0 ml solution #3 (trace elements)
1.0 ml glacial acetic acid
water to 1 liter
For solid medium, add 15 g agar per liter. Autoclave. For Tris-minimal medium omit the acetic acid and titrate the final solution to pH 7.0 with HCl

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