CHAPTER 2 GENERAL MATERIALS AND METHODS
2.8 Preparation of competent E. coli JM 109 cells
Competency of E. coli cells is defined as the ability of the cells to take up foreign DNA and it is expressed as cfu (colony forming unit) per µg DNA. There are two methods for transforming bacterial cells: chemical transformation where bacterial cells are treated with divalent ions (Ca2+) to make them competent, and by electroporation whereby the bacterial cells are exposed to an electric field to create pores through which DNA enters (Dagert and Ehrlich, 1979). Both methods of transformation were used in the present study. Competent E.
coli cells were obtained from Promega Corporation [pGEM®-T Easy Vector System II and Invitrogen Life Technologies (Table 2.1)]. Due to the high cost involved in purchasing chemically competent cells from manufacturers, these were prepared using several methods i.e. The TranformAid™ Bacterial transformation kit from Fermentas life sciences, calcium chloride and a modified TB calcium chloride method (Inoue et al., 1990). Competent cells prepared by TranformAid™ Bacterial transformation kit were very good for transformation, while those using the calcium chloride method were less competent. Although the modified calcium chloride method provided cells with high competence, commercial competent cells were preferred where large numbers of clones were required.
2.8.1 Preparation of competent E. coli JM 109 cells using the TransformAid™
Bacterial Transformation kit 2.8.1.1 Materials
Ampicillin [50 mg/ml]. Ampicillin (0.5 g) was dissolved in dH2O (10 ml), filtered through 0.45 µm filters and stored in a 15 ml conical centrifuge tube at 4°C.
Ampicillin [100 mg/ml]. Ampicillin (1.0 g) was dissolved in dH2O (10 ml), filtered through 0.45 µm filters and stored in a 15ml conical centrifuge tube at 4°C.
2× YT medium. Bacto-tryptone (16.0 g), yeast extract (10.0 g) and NaCl (5.0 g) were dissolved by shaking in dH2O (900 ml). The pH was adjusted to 7.0 with NaOH, and made up to 1000 ml with dH2O. Half of the medium was sealed and autoclaved (121ºC, 30 min, RT) for preparation of bacterial cultures. The remaining half was used for preparation of 2×
YT-ampicillin agar plates.
2× YT-ampicillin agar plates. Agar (7.5 g) was dissolved in 2× YT media (500 ml) and autoclaved (121ºC, 30 min, RT), cooled to 50°C before adding ampicillin to a final concentration of 100 µg/ml. Agar (30-35 ml) was in poured into 85 mm Petri dishes and allowed to solidify.
Glycerol stocks of competent E. coli JM 109 cells
Sterile glycerol [10% (v/v)] Glycerol (10 ml) was made up to 100 ml with sterile dH20, autoclaved (121ºC, 30 min, RT) and kept at 4°C after cooling.
2.8.1.2 Procedure
Competent E. coli JM 109 cells were prepared using TranformAid™ Bacterial transformation kit according to the manufacturer‟s specification.
2.8.2 Preparation of competent E. coli JM 109 cells using the calcium chloride method 2.8.2.1 Materials
Glycerol stocks of E. coli JM 109 cells
0.1 M calcium chloride CaCl2.2H2O (14.7 g) was dissolved in 900 ml of dH2O, the volume adjusted to 1000 ml and autoclaved (121ºC, 30 min, RT).
0.1 M magnesium chloride. MgCl2.6H2O (20.3 g) was dissolved in 900 ml of dH2O, the volumes adjusted to 1000 ml and autoclaved (121ºC, 30 min, RT).
2.8.2.2 Procedure
A fresh colony of E. coli JM 109 cells was inoculated into 5 ml of 2× YT and incubated overnight in a shaker at 37°C. Overnight E. coli JM 109 cell culture (1 ml) was placed in 100 ml of fresh 2× YT in a 500 ml conical flask, and incubated at 37°C in shaker until an OD600= 0.4-0.5 was attained. The cells were centrifuged (6000×g, 5 min, 4°C), the medium drained off, the pellet resuspended in 50 ml of 0.1 M ice-cold MgCl2 and incubated on ice for 30 min.
The cells were centrifuged as before, the MgCl2 drained off and the pellet resuspended in 10 ml of ice cold 0.1 M CaCl2. The cells were stored at -70°C after adding sterile 50% (v/v) glycerol to a final concentration of 20% (v/v) or immediately used for transformation.
2.8.3 Preparation of competent E. coli JM 109 cells with modified TB 2.8.3.1 Materials
TB (CaCl2) solution: PIPES (3.021 g), CaCl2.2H2O (2.205 g) and KCl (18.637 g) were dissolved in dH2O (900 ml) and the pH was adjusted to 6.7 with KOH. MnCl2 (10.885 g) was
dissolved in the solution, the volume was made up to 1000 ml of dH2O and filtered through a 0.45 µM membrane. The solution was stored at 4°C.
S.O.C medium: Bacto-tryptone (20 g), Bacto-yeast extract (5 g) and NaCl (0.5 g) were dissolved in dH2O (900 ml). 1 M MgSO4 (10 ml), 1 M MgCl2 (2.5 ml) were added and the pH adjusted to 7 with NaOH and the volume made up to 990 ml with dH2O. The medium was autoclaved (121ºC, 30 min, RT) and filtered. Glucose (2 M, 10 ml) was added after cooling.
S.O.C-ampicillin agar plates. Agar (7.5 g) was dissolved in S.O.C medium (500 ml) and autoclaved (121ºC, 30 min, RT), cooled to 50°C before adding glucose (2 M, 5 ml) and ampicillin to a final concentration of 100 µg/ml. Agar (30-35 ml) was poured into 85 mm Petri dishes and allowed to solidify.
2.8.3.2 Procedure
An overnight culture of E. coli JM 109 cells (100 µl) was inoculated into fresh S.O.C medium (50 ml) and incubated at 37°C for 1 h. The JM 109 E. coli culture was grown in a 37°C shaker until the cells attained an OD600 of 0.5; the culture (20 ml) was transferred into sterile centrifuge tubes and centrifuged (6000×g, 2 min, 4°C). The medium was drained off, cells resuspended in cold TB (CaCl2) solution (10 ml) and incubated on ice for 25 min. This procedure was repeated twice and the final cell pellet resuspended in cold TB CaCl2 solution (2 ml). The cells were stored at -70°C after adding sterile 50% (v/v) glycerol to a final concentration of 20% (v/v) or immediately used for transformation.
2.8.4 Preparation of E. coli JM 109 cells for electroporation 2.8.4.1 Materials
Glycerol stocks of competent E. coli JM 109 cells Sterile glycerol [10% (v/v)] (Section 2.8.1.1) 2.8.4.2 Procedure
A fresh colony of E. coli JM 109 cells was inoculated into 2×YT medium (Section 2.8.1.1) and grown in a shaker overnight at 37°C. The overnight cell culture (50 ml) was diluted into fresh 2× YT medium (500 ml) in a 1000 ml flask, the cells grown for 2-3 h until OD600 = 0.6.
The cells were transferred into sterile 50 ml conical centrifuge tubes and harvested by centrifugation (5000×g, 10 min, 4°C). The medium was poured off, the cell pellet washed in ice-cold sterile water (25 ml), and centrifuged as before. The water was drained carefully and cells washed again in ice-cold sterile water (12.5 ml) and centrifuged as before. The water
was drained off again and the cells were resuspended in ice-cold 10% (v/v) glycerol (6.25 ml). After centrifugation as before, the glycerol was poured off and the cells resuspended in ice-cold 10% (v/v) glycerol (5 ml). Aliquots (100 µl) were used immediately or frozen at - 70°C.