Molecular Cloning/Preparation of competent cell (''E.Coli'')
Depending on the efficiency of transformation required for various cloning procedures, competent cells were made by two different methods.
Preparation of competent E. coli JM109 cells using rubidium chloride
A single colony of E. coli DH5-α, maintained on a fresh LB agar plate was inoculated into 5 ml of LB medium and incubated at 37C with shaking at 200 rpm for 16 hrs. One ml of this overnight culture was inoculated into 35 ml of LB medium and incubated at 37C at 200 rpm till the OD600 reached 0.55. The culture was then chilled on ice for 15 min and the cells were pelleted at 1,500g for 15 min at 4C in an HB 4 rotor in a Sorvall centrifuge. The supernatant was completely drained and the pellet was gently resuspended in 10 ml of ice cold buffer R1 (100 mM RbCl, 50 mM MnCl2.4H20., 30 mM potassium acetate, 10 mM CaCl2.2H20, 15% glycerol) and incubated on ice for 20 min. The cells were then distributed into prechilled microfuge tubes in 100 µl aliquots, flash frozen in liquid nitrogen and stored at 70C. The competent cells prepared by this method generally yielded a transformation efficiency of 5 x 106 to 1 x 107 colonies/µg of pUC18 DNA and were stable upto three months. They were used for most of the routine cloning experiments involving ligations of compatible, cohesive termini.
100 mM RbCl,
50 mM MnCl2.4H20.,
30 mM potassium acetate,
10 mM CaCl2.2H20,
Preparation of ultracompetent cells
Ultracompetent cells were prepared according to the method described here. A single colony of E. coli DH5 α maintained on a fresh LB agar plate was inoculated into 5 ml of LB and incubated at 37C at 200 rpm for 16 hrs. One ml of this overnight culture was inoculated into 100 ml of LB medium and incubated at 18C at 200 rpm, till the OD600 reached 0.6. The culture was chilled on ice and centrifuged at 1,500g for 15 min at 4C to pellet the cells. The cells were then resuspended in 32 ml of ice cold buffer I (10 mM PIPES pH 6.7, 15 mM CaCl2, 250 mM KCl, 55 mM MnCl2) and incubated on ice for 10 min. The centrifugation was repeated and the cells were resuspended in 8 ml of ice cold buffer I containing 7% DMSO, distributed into 100 µl aliquots, flash frozen in liquid nitrogen and stored at 70C. Ultracompetent cells prepared by this method yielded a transformation efficiency of 1 x 108 colonies/µg of pUC18 DNA and were stable for three months at 70C. Ultracompetent cells were used for cloning experiments involving ligation of DNA fragments with blunt ended termini.
10 mM PIPES pH 6.7,
15 mM CaCl2,
250 mM KCl,
55 mM MnCl2
Transformation of competent and ultracompetent E. coli cells by heat shock
Competent cells were removed from the 70C freezer and thawed on ice. Ligated DNA sample (5 µl) was added to the competent cells and mixed gently. The cells were incubated on ice for 30 min, and then subjected to heat shock at 42C for 90 sec. After the heat shock, 400 µl of LB was added to the cells and the tube was incubated at 37C for 1 hr. The cells were then plated on 90 mm LB agar plates containing 100 µg/ml of ampicillin. In cases where the plasmid vector displayed the property of complementation, the cells were plated on LB agar plates containing the appropriate antibiotic, and coated with 40 µl of 20 mg/ml X Gal and 4 µl of 200 mg/ml IPTG. The plates were incubated at 37C for 14 16 hr to enable the transformants to grow.