d. 3 μl of sample prepared in low stringency buffer was added to the spot activated with low stringency buffer and incubated in humid chamber for 30 min. and removed using whatman strips. (same protocol was repeated for the samples prepared in high stringency buffer on spots activated with high stringency buffer). e. Stringent washings were given to each spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH7.0 for 30 sec and removed using Whatman filter paper strips. f. 1-2 μl of SAP matrix was added to each spot and allowed to dry. g. The chip was then placed in the SELDI machine

One set of cell extracts was prepared in low stringency buffer by mixing cell extracts and low stringency buffet in 1:1 ratio and another in high stringency buffer. b. 10 μl of low stringency/high stringency buffer was added to the spots on the chip and incubated in a humid chamber for 5 min. c. Buffer was removed using Whatman strips without touching the spot surface. This step was repeated once

Activation of LSAX (strong anion exchange ) array

Overnight cell culture raised in LB medium was subcultured 1:100 in LB with 20 mM MgCl2. When the A600 reached 0.4-0.6, the culture was centrifuged at 2800g for 5 min at 4 ̊C. To the cell pellet 0.4 volumes of ice-cold TBF-I buffer was added and incubated on ice for 15 min. The cell suspension was centrifuged at 2800g for 5 min at 4 ̊C and the cells recovered were dissolved in 0.04 volume of ice-cold TBF-II buffer and kept on ice for 45 min. 100 μl aliquots of these competent cells were used for transformation using the normal transformation protocol

TBF method for preparation of high competency cells

For routine plasmid transformations, where high efficiency is not required, the following method which is a modification of that described by Sambrook and Russell (2001) was used. An overnight culture of the recipient strain was subcultured in fresh LB and grown till mid-exponential phase. The culture was chilled on ice for 15 min, and the steps hereafter were done on ice or at 4°C. The culture was centrifuged, and the pellet was resuspended in one third volume of cold 0.1 M CaCl2. After 15 min incubation on ice, the cells were again recovered by centrifugation, and resuspended in one tenth volume of cold 0.1 M CaCl2. The suspension (0.1 ml) was incubated on ice for 1 h after which DNA was added (~10-100 ng of DNA in less than 10 μl volume). The mixture was again incubated on ice for 30 min, and then heat shocked for 90 seconds at 42°C. Immediately 0.9 ml of LB broth was added to the tube and incubated at 37°C for 45 min for phenotypic expression of the antibiotic marker before being plated on selective medium at various dilutions. A negative control tube (with no plasmid DNA addition) was also routinely included in each of the experiments

Calcium chloride method

Transformation protocols