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How To Make Lb Broth. For LB agar add agar to a final concentration of 15. Adjust the final volume of the solution to 1 L with H 2 O. Use the graduated cylinder to measure 500mL of MilliQ water. Seal the cap of the media bottle and shake until components are completely dissolved.
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Add 1 mg ampicillin to 10ml of lb broth. Gibco LB Broth is based on the Lennox formulation one of the most commonly used for LB medium. Example of nutrient agar in a petri dish. Weigh out 20g of premix LB Agar powder VWR DF0445-17 or. In a 1L autoclave bottle orange cap add. Mix powder well to bring into solution 8.
To make about 40 ampicillin plates use the following recipe.
Making the LB Agar 1. Add 1 ul of this stock per 1 ml of autoclaved LB broth or agar after both have cooled below 55C. Add 1 mg ampicillin to 10ml of lb broth. Sterilize by autoclaving for 20 min at 15 psi 105 kgcm 2 on liquid cycle. To make fewer plates use half of all ingredients. While your samples are sterilizing in the autoclave you should prepare your plate pouring station.
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Replace the cap to the bottle but leave it slightly loose for pressure equalization to occur. Make it in 50 ethanol and store it at -20 where it will not freeze. Combine the reagents and shake until the solutes have dissolved. Add 1 ul of this stock per 1 ml of autoclaved LB broth or agar after both have cooled below 55C. Cover the top of the flasks with aluminum foil and label with autoclave tape.
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Making LB Agar Plates Batch makes about 40 plates. Adjust the final volume of the solution to 1 L with H 2 O. 5 g SELECT Yeast Extract. Add dH2O to total volume of 500 mL and transfer to 1 L flask 9. Seal the cap of the media bottle and shake until components are completely dissolved.
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Add 1 ul of this stock per 1 ml of autoclaved LB broth or agar after both have cooled below 55C. Preparation of LB liquid medium. The contents do not have to be completely in solution but any powder left on the sides of the flask will caramelize on the glass during autoclaving. 5 g Sodium Chloride. LB Luria-Bertani liquid medium.
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LB Variations Variations of the LB recipe can be found in the literature and lab manuals. Each bottle of Gibco LB Broth is filter-sterilized and ready-to-use in ergonomic Gibco media bottles with angled openings for simplified pipetting. Carefully pour the measured LB broth powder into the 500mL bottle. Making the LB Agar 1. Heat the mixture to boiling to dissolve agar and sterilize by autoclaving at 15 psi from 121-124C for 15 minutes.
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75 g agar 7. 5 g Sodium Chloride. Spray down the bench with a 70 ethanol solution and wipe down with a paper towel. Formulation per one liter. For Bottle 1 use the recipe above for LB Broth except the final volume should be 500 ml instead of a liter.
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To make about 40 ampicillin plates use the following recipe. 10 g SELECT Peptone 140. Combine the reagents and shake until the solutes have dissolved. 50 g tryptone 4. Adjust the pH to 70 with 5 N NaOH 02 mL.
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Approximately 1mL of 1N NaOH should be expected. Autoclave together with Bottle 2 on liquids cycle for 20 minutes as usual. Cover the top of the flasks with aluminum foil and label with autoclave tape. Combine the reagents and shake until the solutes have dissolved. 5 g Sodium Chloride.
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10 g SELECT Peptone 140. 25g LB broth powder 1000mL ultrapure water Swirl to mix. Example of nutrient agar in a petri dish. Add dH2O to total volume of 500 mL and transfer to 1 L flask 9. Cover the top of the flasks with aluminum foil and label with autoclave tape.
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To make fewer plates use half of all ingredients. Nutrient agar CM0003B and nutrient broth CM0001B from Oxoid share almost the same medium composition. Add 250 mL of dH2O to a graduated cyclindar. While your samples are sterilizing in the autoclave you should prepare your plate pouring station. Use the graduated cylinder to measure 500mL of MilliQ water.
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Mix powder well to bring into solution 8. Formulation per one liter. Autoclave together with Bottle 2 on liquids cycle for 20 minutes as usual. Strictly speaking LB agar should be call ed LA. For Bottle 1 use the recipe above for LB Broth except the final volume should be 500 ml instead of a liter.
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50 g NaCl 6. Add 250 mL of dH2O to a graduated cyclindar. Heat the mixture to boiling to dissolve agar and sterilize by autoclaving at 15 psi from 121-124C for 15 minutes. Make it in 50 ethanol and store it at -20 where it will not freeze. While your samples are sterilizing in the autoclave you should prepare your plate pouring station.
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Make it in 50 ethanol and store it at -20 where it will not freeze. Count out the appropriate number of plates and stack them on your lab bench. The contents do not have to be completely in solution but any powder left on the sides of the flask will caramelize on the glass during autoclaving. Add 250 mL of dH2O to a graduated cyclindar. Neutralize pH 70 the solution using NaOH.
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Place magnetic stirrer into the bottom of the flasks and stir to mix. 50 g NaCl 6. Sterilize by autoclaving for 20 min at 15 psi 105 kgcm 2 on liquid cycle. Powder will not dissolve completely that is ok. The contents do not have to be completely in solution but any powder left on the sides of the flask will caramelize on the glass during autoclaving.
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Formulation per one liter. Mix powder well to bring into solution 8. Replace the cap to the bottle but leave it slightly loose for pressure equalization to occur. Add dH2O to total volume of 500 mL and transfer to 1 L flask 9. Powder will not dissolve completely that is ok.
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Add 1 ul of this stock per 1 ml of autoclaved LB broth or agar after both have cooled below 55C. 50 g tryptone 4. Replace the cap to the bottle but leave it slightly loose for pressure equalization to occur. 5 g Sodium Chloride. Place magnetic stirrer into the bottom of the flasks and stir to mix.
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5 g SELECT Yeast Extract. Powder will not dissolve completely that is ok. Add 250 mL of dH2O to a graduated cyclindar. Add 1 mg ampicillin to 10ml of lb broth. To make about 40 ampicillin plates use the following recipe.
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Approximately 1mL of 1N NaOH should be expected. Approximately 1mL of 1N NaOH should be expected. The usual approach is to make a stock solution of ampicillin at 100 mgml. Example of nutrient agar in a petri dish. 75 g agar 7.
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Preparation of LB liquid medium. Making the LB Agar 1. 50 g NaCl 6. 5 g Sodium Chloride. Replace the cap to the bottle but leave it slightly loose for pressure equalization to occur.
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