Methods with bacteria

 

E. coli Overnight Culture

Solutions:

LB (Luria bretani) broth: 10 g tryptone, 5 g yeast extract, 10 g NaCl, in 1 L AD, pH 7.0. Sterilization by autoclaving

Antibiotic stock solutions 1000x: ampicillin 100 mg/mL, kanamycin 25 mg/mL.

For preparing a saturated E. coli over night culture, 5 mL LB broth containing antibiotics are inoculated with a single colony of the desired bacteria strain in a 50 mL tube. Incubation is carried out at 37°C with constant shaking at 200 rpm over night (o/n). After 12 hours the culture is in the state of stationary phase, no more increase of biomass is observed.

 

Preparation of Electro-competent E. coli

Solutions:

LB broth
AD sterile, 4°C
10 % glycerol, sterile filtered, 4°C
liquid N2

10 mL LB broth are inoculated with E. coli HB101 and an o/n culture are prepared. On the next day the o/n culture is diluted in 1 L LB broth and incubation is continued for around 3 hours. Optical density (OD) at 600 nm is measured after 1 hour respectively until OD 0.4 - 0.6 is reached. By that means the bacteria are in the state of exponential phase. The bacteria broth is then cooled down on ice for 15 – 30 minutes in order to stop the bacteria from dividing. Harvesting of bacteria is carried out by centrifugation at 2500 x g, 4°C for 15 minutes (centrifuge: Sorvall RC-5B), the supernatant is removed and collected for autoclaving. The bacteria pellet is resuspended in 1 L sterile AD (4°C) and again centrifuged at 2500 x g for 15 minutes at 4°C. The supernatant is discarded and the pellet is resuspended in 500 mL sterile AD (4°C) and again centrifuged at 2500 x g for 15 minutes at 4°C. The supernatant is discarded and the pellet is resupended in 20 mL 10 % sterile glycerol (4°C) and again centrifuged at 2500 x g for 15 minutes at 4°C. The supernatant is discarded and the pellet is resuspended in 2 - 5 mL 10 % sterile glycerol (4°C). 50 µL aliquots of this bacteria solution are prepared and quickly frozen in liquid N2. Aliquots are stored at -80°C and can be used for 1 year.

 

Electroporation Transformation of E. coli

Solutions:

SOC medium for recovery: LB broth containing glucose 20 mM, MgSO4 10 mM, MgCl2 10 mM

 

50 µL electro-competent bacteria aliquot is thawed on ice and transferred into prechilled electroporation cuvettes, 0.5 - 1 µg plasmid DNA (or 5 - 10 µL of ligation mix) is added (0.1 cm). The surface of the cuvette was dried prior to electroporation. Electroporation parameters (power supply : voltage: 1.25 – 1.9 kV, capacity: 25 µF, resistance: 200 Ω. A successful electroporation is achieved with a time constant between 4 – 5 ms. After electroporation the bacteria are resuspended in 1 mL of SOC medium and transferred in a 3059 Falcon tube. The bacteria are then incubated at 37°C at 200 rpm for 1 hour. After recovery the transformed bacteria are briefly centrifuged, resuspended in 100 µL SOC medium and the suspension is plated on LB agar plates containing appropriate antibiotics. The plates are incubated over night at 37°C.

 

Preparation of Heat-Shock Competent E. coli

Solutions:

LB broth

TSS Buffer: 1 % Bacto-Trypton, 0.5 % yeast extract, 0.5 % NaCl, 5 % DMSO (dimethylsulfoxide), MgCl2 50 mM, pH 6.5, 10% PEG (MW 3000, 3350), fill up with AD, sterile filtered, stored at 4°C.

Glycerol 87 %

Liquid N2

10 mL LB broth are inoculated with E. coli HB101 or another appropriate strain such as DH5a and an o/n culture is prepared. On the next day the o/n culture is diluted in 1 L LB broth and incubation is continued for around 3 hours. Optical density (OD) at 600 nm is measured after 1 hour respectively until OD 0.4 – 0.6 is reached indicating that the bacteria are in the exponential phase of growth. The bacteria broth is then cooled down on ice for 15 – 30 minutes in order to stop the bacteria from dividing. Harvesting of bacteria is carried out by centrifugation at 2500 x g, 4°C for 15 minutes (centrifuge: Sorvall RC-5B). The supernatant is removed and collected for autoclaving. The pellet is resuspended in a total volume of ~ 50 mL (36 mL TSS buffer + 12 mL of glycerol, 1/20 volume of diluted culture. Aliquots of 200 µL are prepared, quickly frozen in liquid N2 and stored at -80°C.

 

Heat Shock Transformation of E. coli

Solutions:

SOC medium: LB broth containing 0.36% glucose, MgSO4 10 mM, MgCl2 10 mM

An aliquot of 50 µL heat shock competent E. coli (DH5a, stored at –80°C) is quickly thawed in the palm and left for 10 minutes on ice. The aliquot is then transferred into a pre-chilled Falcon 2059 tube. About 1 µg of DNA (1 - 2.5 µL) is pipetted into the competent bacteria and the tube is gently swirled. After 20 minutes of incubation on ice the tube is placed in a prewarmed 42°C water bath for 90 seconds without moving the tube. In order to cool down the sample, the tube is immediately placed on ice for 1-2 minutes. Thereafter 800 µL of SOC medium are added to the transformation mix and the bacteria are incubated for 1 hour at 37°C and 200 rpm to allow recovery from the heat shock and start expression of the selection gene. Plating: For simple retransformations 100 µL are plated on appropriate LB (Luria Bertani) agar plates (prewarmed to 37°C) supplemented with the appropriate antibiotics (kanamycin 25 µg/mL or ampicillin 100 µg/mL). For clonings after a ligation the whole bacterial suspension is used by pelleting the bacteria of the transformation mix by briefly and gently spinning down, discarding the supernatant and resupension of the pellet in 100 µL SOC medium (or the reminder of the supernatant). This concentrated suspension containing all the bacteria is then plated on LB agar plates containing appropriate antibiotics. The plates are incubated over night at 37°C.

 

Glycerol Stock of E. coli

Solutions:

Glycerol 87 %

For a -80°C E. coli glycerol stock, 400 µL of an E. coli o/n culture are mixed thoroughly with 100 µL 87 % glycerol in a 1.5 ml reaction tube and stored at -80°C. For inoculation of fresh LB broth, a yellow 200 µL tip is plunged into the frozen (!) stock and then pipetted up and down in the LB broth in order to thaw and resuspend the bacteria.