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Stock Reagent Recipes

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Stock Reagent Recipes

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    1. Compiled By: Laurie Brier Stock Reagent Recipes

    2. Table of contents

    3. Table of Contents (Cont.)

    4. Table of Contents (Cont.)

    5. S Basal Medium Start with a bottle of 500 mL of distilled water. Add: 2.93 g of NaCl 0.5 g of K2HPO4 3 g of KH2PO4 500 無 of Cholesterol Autoclave the solution. Makes 500 mL.

    6. 1 M Anhydrous CaCl2 Dissolve 11.099 g of CaCl2 into 100 mL of distilled water. Makes 100 mL

    7. Cholesterol Autoclave 250 mL bottles. Use a graduated cylinder to pour 50 mL of 95% ETOH into a sterile falcon tube. Also, measure 0.25 g of cholesterol into a clean weigh boat.

    8. Cholesterol (Cont.) Pour the ETOH into a 250 ml bottle and add the cholesterol. Close the bottle and shake to mix. Make sure the solution is clear or heat the solution to make sure the cholesterol is in solution. Makes 50 mL.

    9. 0.5 Gelatin Place 0.5 g of dry, solid gelatin 100 mL of distilled water. Autoclave the mixture. Makes 100 mL.

    10. 30% Glycerin Mix 30 mL of glycerin with 70 mL of distilled water. Autoclave the solution. Makes 100 mL

    11. 500 mM KCl Dissolve 3.728 g of KCl into 100 mL of distilled water. Autoclave the solution. Makes 100 mL.

    12. 1 M KH2PO4 (pH 6) Dissolve 17.418g KH2PO4 into 100 mL of distilled water. Use 5 M NaOH to bring the pH to 6. Makes 100 mL

    13. 25 mM MgCl Dissolve 0.508 g of MgCl into 100 mL of distilled water. Autoclave the solution. Makes 100 mL.

    14. 1 M MgSO4 Dissolve 12.037g MgSO4 into 100mL of water. Makes 100 mL

    15. 1 M NaCl Dissolve 5.844 g of NaCl into 100 mL of water. Makes 100 mL

    16. NaOH Solutions Use 100 mL of distilled water. For 1 N NaOH, add 4 g of NaOH. For 10 N NaOH, add 40 g of NaOH. Use caution. The solution could get very hot! Makes 100 mL.

    17. PCR Lysis Buffer Into a small, sterile tube mix: Pro K (at 1 mg/ml) ? 3 無 100 mM Tris ? 100 無 500 mM KCl ? 100 無 25 mM MgCl2 ?100 無 4.5% Tween ? 100 無 0.5% Gelatin ? 100 無 Distilled water ? 497 無 Makes 1 mL

    18. Pro K Mix 2 無 of stock Pro K (20 mg/ml) with 38 無 of distilled water. The Pro K is now at a concentration of 1 mg/ml Aliquot this new diluted Pro K solution into sterile microtubes.

    19. 1 X TBE Dilute 100 mL of 10 X TBE with 900 mL of distilled water Makes 1 Liter

    20. 100 mM Tris Dissolve 1.214 g of Tris into 100 mL of distilled water. Autoclave the solution. Makes 100 mL.

    21. 4.5% Tween Dissolve 4.5 mL of Tween 20 into 95.5 mL of distilled water. Autoclave the solution Makes 100 mL.

    22. 2X YTB Into an empty bottle add: 4 g of tryptone 2.5 g of yeast extract 1.25 g of NaCl Then add 250 mL of distilled water. Autoclave the solution. Place 10 mL of the autoclaved solution into each of 25 sterile test tubes.

    23. Inoculating OP-50 bacteria into 2X YTB Place 10 ml of 2X YTB into a sterile 15 ml tube Fetch a microtube of OP-50 bacteria from the freezer. Using the aseptic technique, inoculate the 2X YTB buffer with a loopful of bacteria. Leave the tubes in an incubator (37 C) overnight.

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