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sihuanchen

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[交流] 【求助/交流】5-氟乳清酸培养基已有2人参与

5-氟乳清酸培养基配制，是先加5-氟乳清酸再灭菌还是灭菌后再加？

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1楼 2010-05-16 15:38:46

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sihuanchen(金币+1):谢谢参与

过滤灭菌，然后加到灭过的培养基中

[ Last edited by lingn on 2010-5-16 at 20:11 ]

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2楼2010-05-16 20:06:41

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amisking(金币+3):good！ 2010-05-16 21:52:28
sihuanchen(金币+20): 2010-08-24 17:08:43

琼脂培养基先灭菌冷却到50-60度时再加入用0.2 um滤膜过滤除菌的5-氟乳清酸，提醒一下，5-氟乳清酸再水里，甲、乙醇都很难溶解，在水里溶解要加热到50度左右搅拌一个小时左右才充分溶解，溶解后用0.2 um滤膜过滤除菌。我把我用的protocol给您贴出来吧！
Good luck with your experiments!

5-FOA (5-Fluoroorotic Acid) Medium and Agar Plate Recipe
Following recipe makes 1 liter 5-FOA plates:

Step-1: Autoclave 20 grams of agar in 750 ml double distilled water.
Step-2: Add followings into a glass beaker, and bring to 250 ml with double distilled water.

Ammonium Sulfate                               5.0 g
Yeast nitrogen base without amino acid  1.7 g
Dextrose                                              20 g
Necessary nutritional supplements (see note 3)  X g
5-FOA                                                    1.0 g

Warm up 5-FOA mixture in Step-2 at 55-65°C on a heat plate, stir until it dissolves ~30 min-1 h. The solubility of 5-FOA is very low in water; therefore, it is necessary to warm up the solution. Some manufacturers, i.e., US Biological, claim that 5-FOA is resistant to autoclaving. I did not try to autoclave it myself, but you can try it.

After 5-FOA containing mixture in Step-2 is completely dissolved, sterilize it by filter. Place both melted agar and 5-FOA containing solution in a water bath (50-60°C) for one hour. Finally, add 5-FOA solution onto melted agar and pour your plates. Plates may be dried at RT for 48 hours and may be kept at 4°C for long term storage.

Notes:

1.Do not forget to add Uracil into the plates at 20 mg/L of final concentration (Do not add too much Uracil!).
2.Never adjust the pH of 5-FOA containing medium. Only protonated form of 5-FOA is permeable. pH of your plates will be very acidic after adding 5-FOA.
3.Do not add more nutritional supplements than it is necessary; this reduces efficacy of 5-FOA plates. For example, you only need to add His, Leu, Lys and Ura supplements for strain BY4742. These supplements may be added directly from 100x stock solution.
4.5-FOA is very expensive reagent. I found that US Biological has the cheapest price for 5-FOA.
5.5-FOA is highly mutagenic to Candida albicans. This may also be the case for budding yeast. I suggest you do not leave yeast colonies on 5-FOA plates for longer than it is necessary. Wellington and Rustchenko, Yeast, 2005, Volume 22, Issue 1, Pages 57-70).

Other sources of 5-FOA is Zymo Research and Sigma. The US Biological sells the cheapest 5-FOA reagent. I do not know if purity of 5-FOA is different from each company. 5-FOA from US Biological worked great for me. Do not waste your money!

Good luck with your experiments!

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3楼2010-05-16 20:21:30

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