Analysis of Transformation Results

Plates should be collected from the cold-room and colonies on each should be examined. Colonies of an unusual morphology and color may indicate contamination of competent cells of E. coli. The type and number of the colony on each plate should be determined. The type indicates whether it exhibits a positive or negative GAP. With the observation of large colonies, counting colonies has to be simplified with the division of plates into quadrants. The exhibition of GAP can be determined based on color. Although cells of E-coli are generally green, colonies which express GAP can be green if GAP levels are significantly high. The expression of GAP can be confirmed by analyzing fluorescence under the UV light.

1.    Inoculation of Overnight Culture

Throughout this procedure, the aseptic technique should be used. Four overnight cell cultures should be set up, two from cell cultures from colonies which don’t product GAP and two from the ones which produce GAP. Four tubes should be collected, labelled with bench numbers and initials, and –GAP should be labeled on one while +GAP on the other. Culture plates should be collected and +GAP tubes should be inoculated with cells of colonies which produce GAP from plate 1, and –GAP tubes with different cells from colonies which don’t produce GAP from plate 2.

2.   Induction of the Synthesis in Transformed Cells

Two tubes containing overnight cultures of E. coli should be collected that was set up the day before. A sterile 250 ml flask should be collected and a 50 ml of LB media should be added. The growth medium is per-warmed to almost 37 Celsius for enhancing the initial cell growth rate. Sufficient stock of ampicillin should be added to LB for giving a final 100 g/ml concentration