Category: Reagents
Preparation of SOB (Super Optimal Broth) Medium
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.
Preparation of 10 M Sodium Hydroxide (NaOH) Solution
Here we have described a procedure to prepare 10 M (or 10 N) sodium hydroxide (NaOH) solution. A 10 M NaOH solution can be prepared by dissolving 40 g of NaOH (Molecular Weight: 40.00) in water to a final volume of 100 ml. Since dissolution of NaOH in water is a highly exothermic reaction, extreme care must be taken while preparing the solution.
Preparation of 5X TBE Electrophoresis Buffer
TBE is a very common electrophoresis buffer for DNA agarose gel electrophoresis. It is prepared as a 5X stock solution that can be diluted to 1x or 0.5x working solution for DNA agarose gel electrophoresis. The 5X concentrated solution contains 0.445 M Tris borate and 0.01 M EDTA (pH 8.2 – 8.4).
A 5X stock solution is prepared by dissolving 54 g Tris base, 27.5 g Boric acid, and 20 ml of 0.5 M EDTA in water to a final volume of 1000 ml. The solution is sterilized by autoclaving and is stored at room temperature.