Biology Protocols

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Terrific Broth

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Terrific Broth contains slightly more Bacto® Tryptone for amino acids than LB, but nearly 5x the Bacto® Yeast Extract, supplying large quantities of growth factors and nucleic acid precursors. TB also contains phosphate buffer to maintain a physiological pH during exponential growth and glycerol (which is not metabolized into lactic acid) at a non-rate-limiting concentration. Bacteria grow longer and lyse more slowly, increasing the amount of plasmid DNA recovered. Buffering tends to be needed if the media contains a carbon source, like glucose, which is turned into organic acids (acetate) in lieu of aerobic metabolism. Or if the carbon has to be rested from a typical nitrogen source, like protein (as in the case of Super Broth) which results in a pH rise as amines are stripped. Tris is a good buffer but typically a phosphate buffer system is used (although PIPES and MOPS are used in minimal media formulations). Giving a more readily available carbon source, like glycerol, would also enhance the media.

One litre final volume:

For 900ml of YTG base:

Measure 900ml of distilled H2O.

Add 12g Bacto Tryptone.

Add 24g Bacto Yeast Extract.

Add 4ml glycerol.

In a separate flash:

Measure 90ml of distilled H2O.

Add 2.31 g KH2PO4 monobasic .

Add 12.54 g K2HPO4 dibasic (for trihydrate 16.45 g)

Adjust volume to 100 mL with dH2O

Sterilize each solution separately volume by autoclaving. Solutions should only be combined after the YTG base cools to 60oC.

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