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Medium Preparation

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Medium Preparation

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    1. Medium Preparation Composition of MS medium Discussion of ingredients and what they are for Use of prepackaged mixes or stock solutions How stocks are used to make medium Preparation of stocks

    2. Murashige-Skoog Medium Composition

    3. Murashige-Skoog Medium Composition

    4. Medium ingredients Inorganics macronutrient salts micronutrient salts Organics sugar (sucrose) "vitamins" plant growth regulators (PGRs) undefined ingredients agar and solidifying agents

    5. Prepackaged mixes vs. medium from stocks Prepackaged mixes offer convenience Stock solutions have several benefits the assurance that you know precisely what is in the medium the potential to modify the medium; e.g., to adjust the PGR concentrations and ratios the assurance that all of the chemicals are in solution

    6. Use of prepackaged mixes Most packaged mixes come with directions There are often many variations of the most commonly used media The most widely used come with salts and PGRs for 1 L, which are dissolved in water Sucrose is added, pH checked and adjusted if necessary

    7. Use of prepackaged mixes (contin) Solidifying agent is added Medium is autoclaved and allowed to cool

    8. Use of stock solutions For making a liter of MS medium, start with 700 to 800 ml DI H2O in a beaker Place a stir bar in the beaker, and stir while adding 30 g sucrose Add inorganic and organic stock solutions: Nitrate stock (50x) 20 ml Sulfate stock (100x) 10 ml Halide stock (100x) 10 ml

    9. Use of stock solutions (contin) Add inorganic and organic stocks: P•B•Mo (100x) 10 ml Na2FeEDTA (200x) 5 ml myo-inositol (100x) 10 ml vitamins (1000x) 1 ml PGRs (usu. 1 mg/ml) 0-5 ml Bring volume to near 1000 ml with DI water

    10. Use of stock solutions (contin) Adjust the pH to 5.7 (most species) Bring the volume of the medium to 1 L using a graduated cylinder Add 8 grams agar Place beaker in the microwave to melt the agar While medium is still warm (above 40° C), pour about 35 ml into each baby-food jar Loosely cap jars and autoclave 15 min at 121° C

    11. Preparation and use of stock solutions Stocks are usually made as 50x, 100x, 200x or 1000x the medium concentration For example, for a nitrate stock at 50x, the medium recipe calls for 1650 mg/L of NH4NO3, you would make a stock at a conc. of 50(1650) = 82,500 mg/L or 82.5 g/L It may be more convenient to make a smaller volume of stock solution, say 200 ml of nitrate stock

    12. Preparation and use of stock solutions (contin) How would that change the mass (no. grams) of NH4NO3 added to the stock? Remember, you always want the correct conc. of each ingredient in the medium One way to keep track of this is with the formula VsCs = VmCm, where: Vs = volume of the stock solution Cs = concentration of the stock solution

    13. Preparation and use of stock solutions (contin) Vm = volume of the culture medium Cm = concentration of the culture medium This formula also comes in handy in calculating the volume of stock to add: if the conc. of the stock is 82.5 g NH4NO3 and you are making 1 liter of medium, the stock volume is x(82.5 g) = (1000 ml)(1650 mg/L), and solving for x, the stock volume is (?) does your answer agree with the volume listed on slide 8?

    14. Making PGR stocks A useful concentration for PGRs is 1 mg/ml (1000 mg/L), then, e.g., 2 mg/L BA can easily be made up by dispensing 2 ml of a 1 mg/ml BA stock What if you were making only 250 ml of medium?

    15. Making PGR stocks (contin) For making PGR stocks, keep in mind that these go into aqueous solution with difficulty One trick is to recall that most PGRs are weakly acidic or basic; e.g., cytokinins are weak bases, auxins are weak acids Using sl. excess of molar equivalents of the opposing compound allows most PGRs to be completely dissolved

    16. Making PGR stocks (contin) For example, for making 1 mg/ml stock of BA, dissolve 100 mg BA in 98.6 ml DI water and 1.4 ml 1 N HCl Solution is stirred until all granules go into solution, which may take several hours For auxins, use 1 N NaOH or KOH in place of the acid

    17. Considerations in medium prep Stock solutions should be stored at 4° C to retard bacterial and fungal growth Inorganic stocks can be sterilized; when dispensing from sterile stocks, use sterile pipets Some organic compounds must be filter-sterilized Vitamins and some PGRs can be autoclaved Exceptions: IAA, GA, ABA Antibiotics must be filter-sterilized

    18. Considerations in medium prep (contin) Water of crystallization check your reagent against the medium recipe; a molecule of ZnSO4 and ZnSO4?7H20 weigh different amounts using molecular weights, calculate the amount that is proportional to the compound cited in the recipe Recommended shelf life of media and stocks for medium and stocks, about 2 months for medium with antibiotics, use ASAP for stable PGRs, up to 6 months

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