LB Broth

From 2011.igem.org

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(LB Recipe)
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'''Luria-Bertani Media (LB Broth)'''
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under construction -- feel free to finish this  DrLiz
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  Tryptone        1.0%    (a proteolytic digest of milk proteins)
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  Yeast Extract  0.5%    (a source of vitamins and nutrients)
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  NaCl            1.0%    (table salt - guess those little E. coli's like tasty food just like people do)
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Note: One ml of water weighs 1 gram (under ideal conditions -- for us it's close enough). It is by weight that we measure the dry ingredients.
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So, when you wish to have x% of some ingredient in your final solution, the calculation looks like this:
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  For each 100 ml final solution, use x grams of the ingredient.
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So, if we wish to make 1 liter (1000 ml) of LB Broth, we measure the following amounts of the dry ingredients:
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10 gr Tryptone
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  5 gr Yeast Extract
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  1 gr NaCl
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Add some of the water and adjust the pH using 10 N NaOH (about 0.1 ml)
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Then add enough water to have a final volume of 1 liter. Use a large flask with measuring lines - the volume will be close enough.
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Next use a funnel to aliquot about 200-250 ml into clean bottles that will serve as storage bottles, and autoclave on liquids cycle for 20 minutes at 15 psi (121 degress Centigrade is normal).
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==With Ampicillin==
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If you are going to add antibiotic, wait until the broth has cooled enough that you can handle the bottles. To each 1 ml broth, add 1 ul of 50 mg/ml ampicillin. (So, for 200 ml bottle, add 200 ul ampicillin).
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Put into the refridgerator for storage.
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==For Agar Plates==
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To make about 40 ampicillin plates use the following recipe. To make fewer plates, use half of all ingredients.
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'''Bottle 1'''
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For Bottle 1, use the recipe above for LB Broth, '''except the final volume should be 500 ml''' instead of a liter. Autoclave together with Bottle 2 on liquids cycle for 20 minutes, as usual. Do not add ampicillin until the bottles have cooled.
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'''Bottle 2'''
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20 gr Bacto agar
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500 ml water
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After autoclaving, let them cool enough to be handleable. Add 1000 ul of 50 mg/ml ampicillin to Bottle 1. Mix by swirling.
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Add Bottle 2 to Bottle 1 and mix by gentle inversion. Loosen cap and let it sit for enough minutes that the bubbles rise to the top.
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Label bottoms of the plates with "Amp - LB", and gently pour about 20-25 ml of the liquid agar in each plate one-by-ove, covering every plate immediately with its top plate before pouring the next. Gently swirl each plate right after you've poured it, so the bubbles are distributed around the edges.
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Let them sit on the bench several hours or overnight, then transfer to -4 degrees Centigrade for storage.

Latest revision as of 01:22, 18 August 2011

Luria-Bertani Media (LB Broth) 

 Tryptone        1.0%     (a proteolytic digest of milk proteins)
 Yeast Extract   0.5%     (a source of vitamins and nutrients)
 NaCl            1.0%     (table salt - guess those little E. coli's like tasty food just like people do)

Note: One ml of water weighs 1 gram (under ideal conditions -- for us it's close enough). It is by weight that we measure the dry ingredients.

So, when you wish to have x% of some ingredient in your final solution, the calculation looks like this: 

For each 100 ml final solution, use x grams of the ingredient.

So, if we wish to make 1 liter (1000 ml) of LB Broth, we measure the following amounts of the dry ingredients: 

10 gr Tryptone 
 5 gr Yeast Extract
 1 gr NaCl

Add some of the water and adjust the pH using 10 N NaOH (about 0.1 ml)

Then add enough water to have a final volume of 1 liter. Use a large flask with measuring lines - the volume will be close enough.

Next use a funnel to aliquot about 200-250 ml into clean bottles that will serve as storage bottles, and autoclave on liquids cycle for 20 minutes at 15 psi (121 degress Centigrade is normal).

With Ampicillin

If you are going to add antibiotic, wait until the broth has cooled enough that you can handle the bottles. To each 1 ml broth, add 1 ul of 50 mg/ml ampicillin. (So, for 200 ml bottle, add 200 ul ampicillin).

Put into the refridgerator for storage.

For Agar Plates

To make about 40 ampicillin plates use the following recipe. To make fewer plates, use half of all ingredients.

Bottle 1

For Bottle 1, use the recipe above for LB Broth, except the final volume should be 500 ml instead of a liter. Autoclave together with Bottle 2 on liquids cycle for 20 minutes, as usual. Do not add ampicillin until the bottles have cooled.

Bottle 2 

20 gr Bacto agar
500 ml water

After autoclaving, let them cool enough to be handleable. Add 1000 ul of 50 mg/ml ampicillin to Bottle 1. Mix by swirling.

Add Bottle 2 to Bottle 1 and mix by gentle inversion. Loosen cap and let it sit for enough minutes that the bubbles rise to the top.

Label bottoms of the plates with "Amp - LB", and gently pour about 20-25 ml of the liquid agar in each plate one-by-ove, covering every plate immediately with its top plate before pouring the next. Gently swirl each plate right after you've poured it, so the bubbles are distributed around the edges.

Let them sit on the bench several hours or overnight, then transfer to -4 degrees Centigrade for storage.

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