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Unformatted text preview: Christopher Wilkerson 1 5/12/09 Making Competent Cells Objective : The purpose of this lab is to grow up a culture of bacteria for later experiments, learn to identify the log phase of bacterial growth, learn how to use a centrifuge, and to make bacterial cells chemically potent. Lab Protocol : Initially a stock of bacterial culture was taken from a 4 C freezer. 500 L of the culture was then transferred, using a micropipette, into a 250 mL stock solution of LB broth at room temperature. Next the broth with culture was placed in an agitating incubator at 37 C. A spectrophotometer was then set up using the Logger Pro system. The spec. was blanked using LB broth without bacteria to ensure accurate readings. Approximately every 20 minutes a 1 mL sample of the bacteria was taken and placed into a cuvette. This was then put into the spectrophotometer. The absorbance was then recorded at 600 nm. This was repeated ~20 minutes until the absorbance reached roughly 0.35. This process took several hours, and in-between absorbance readings preparations were ongoing for bacterial harvest. First a bucket of ice was obtained to chill all components that will come into contact with the bacteria after harvesting the bacteria. Next a 50 mL sterile conical tube, along with 50 mL of 0.1 M CaCl 2 , prepared from 1 M Stock solution, in another 50 mL sterile conical tube were placed on ice in the bucket. 40 1.5 mL eppendorf tubes were sterile conical tube were placed on ice in the bucket....
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