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sihuanchen新虫 (小有名气)
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【求助/交流】5-氟乳清酸培养基 已有2人参与
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| 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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yatou996512
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4楼2011-10-10 16:36:06
lingn
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2楼2010-05-16 20:06:41
Atalpha
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sihuanchen(金币+1):谢谢参与
amisking(金币+3):good！ 2010-05-16 21:52:28
sihuanchen(金币+20): 2010-08-24 17:08:43
sihuanchen(金币+1):谢谢参与
amisking(金币+3):good！ 2010-05-16 21:52:28
sihuanchen(金币+20): 2010-08-24 17:08:43
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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! |
3楼2010-05-16 20:21:30
