Protocol – Growing Large Volume of Liquid Culture of Escherichia coli for Large-Scale Plasmid Isolation
Large-scale isolation of plasmid requires a large volume of E. coli culture. Large scale plasmid isolation procedures are termed, midiprep (25 – 50 ml starting culture volume) and maxiprep (100 – 500 ml starting culture volume). A starter culture is initially prepared by inoculating a colony in a small volume (2 – 10 ml) of culture medium. Large culture volume is prepared by diluting starter culture in a ratio of 1: 100 to 1: 1000 in the growth medium.
Lambda-agar is nothing but a lambda broth which is solidified by agar. Agar is a solidifying agent. It melts at 85°C but melton agar stays liquid at a temperature > 40°C (this property is called hysteresis, different temperatures for melting and solidifying). This allows us to add heat-labile medium supplements such as antibiotics and vitamins and prepare solid medium. Lambda-agar solid medium is prepared by dissolving 15 g agar / liter of Lambda broth.
OVERVIEW TNT medium is a moderately rich medium. It contains tryptone, sodium chloride (NaCl) and thiamine. Tryptone provides basic nutrients…
OVERVIEW M9 minimal medium is a chemically-defined minimal growth medium, used to cultivate Escherichia coli. It is prepared by supplementing…
SOC (Super Optimal broth with Catabolite repression) medium is a nutrient-rich microbial growth medium, formulated by Douglas Hanahan in 1983 (Hanahan, 1983). SOC medium is prepared without Mg++ and Glucose by dissolving 20 g bacto-tryptone, 5 g bacto-yeast extract, 2 ml of 5M sodium chloride, and 2.5 ml of 1M KCl in water to a final volume of ≈ 1000 ml (960 ml). Magnesium salts (i.e., MgSO4 and MgCl2, 10 ml each of 1M MgSO4 and 1M MgCl2) and Glucose (10 ml of 1 M Glucose) are added to the medium after autoclaving to prevent any chemical reactions and precipitation of the medium components, and degradation of Glucose.
LB is a rich growth medium, commonly used to grow E. coli. LB Luria Broth contains 1% Bacto-tryptone, 0.5% Bacto-yeast extract and 0.05% Sodium chloride. A 1000 ml LB Luria Broth is prepared by dissolving 10 g Bacto-tryptone, 5 g Bacto-yeast extract and 0.5 g sodium chloride in deionized water to a final volume of 1000 ml. After adjusting the pH 7.0 with 5N NaOH medium is autoclaved and stored in a cold room.
LB (Lysogeny broth or Luria-Bertani) is the most commonly used growth medium for E. coli culture. LB is a rich medium that contains tryptone, yeast extract and sodium chloride. It promotes fast growth of E. coli in culture by fulfilling all nutritional requirements, thus suitable for all regular molecular biology applications including molecular cloning, plasmid amplification, and recombinant protein production. Several versions of the original recipe formulated by Giuseppi Bertani in 1951 are now in common use: Miller broth, Lennox broth, Luria broth. They differ in NaCl concentration.
SOB (Super Optimal Broth) medium is a nutrient-rich microbial growth medium, designed by Douglas Hanahan in 1983. SOB or its glucose-supplemented version SOC is often used during transformation of E. coli competent cells with plasmids (added in transformed cells after heat shock). It results in better survival and higher transformation efficiency as compared to regular LB medium. It contains 2% Bacto-tryptone, 0.5% Bacto-yeast extract, 0.05% Sodium chloride, 2.5 mM KCl, and 10 mM MgCl2. A 1000 ml SOB medium is prepared by dissolving 20 g bacto-tryptone, 5 g bacto-yeast extract, 0.5 g sodium chloride and 2.5 ml of 1M KCl in water to a final volume of 1000 ml. Medium is sterilized by autoclaving. MgCl2 (5 ml of 2M MgCl2) is added just before use.
Ampicillin Sodium salt can be purchased from any of the following suppliers. You must visit the supplier’s homepage to order…
LB medium is a rich growth medium, commonly used to grow E. coli. LB, Lennox Broth contains 1% Bacto-tryptone, 0.5% Bacto-yeast extract and 0.5% Sodium chloride. A 1000 ml LB, Lennox Broth is prepared by dissolving 10 g Bacto-tryptone, 5 g Bacto-yeast extract and 5 g sodium chloride in deionized water to a final volume of 1000 ml. After adjusting the pH 7.0 with 5N NaOH medium is autoclaved and stored in a cold room.
OVERVIEW Lambda Broth is a moderately rich medium. It is useful in the propagation of λ Phage. It contains tryptone…
Ampicillin is one of the most extensively used antibiotics as a selection marker in molecular biology and bacteriology. Ampicillin is available as Ampicillin anhydrous, Ampicillin trihydrate and Ampicillin sodium salt. Ampicillin sodium salt is often used to prepare stock solution as it has better solubility in water (>50 mg/ml in water). A 50 mg/ml stock solution of ampicillin can be prepared by dissolving 500 mg ampicillin sodium salt in water to a final volume of 10 ml.
The 2 x YT medium, also known as a 2 x TY medium, is a rich medium. It is recommended for the growth and propagation of E. coli infected with the single strand filamentous bacteriophage M13. It can be prepared by dissolving 16 g tryptone, 10 g yeast extract, and 5 g Sodium chloride (NaCl) in water to a final volume of 1000 ml.
Amplification of plasmid is desirable for many applications including gene cloning, DNA sequencing, transfection, and probe preparation. The fastest and routinely used method to amplify plasmid is to introduce plasmid in an appropriate strain of E. coli e.g., DH5α (the process is called transformation), grow them to a suitable culture volume, and finally, extract plasmid from them (the process called plasmid isolation).