9. You perform a transformation experiment with a plasmid that encodes a gene th
ID: 275235 • Letter: 9
Question
9. You perform a transformation experiment with a plasmid that encodes a gene that confers resistance to a drug called kanamycin. a) Describe the controls needed in this experiment to determine if contamination has occurred. b) You forget to incubate the bacteria at 37C for 30 min. Predict the results of your experiment under the following conditions (lots, some, or no growth): LB plate (-) plasmid LB plate (+) plasmid LB+kan plate (-) plasmid LB+kan plate (+) plasmid c) You forget to heat shock the bacteria. Predict the results of your experiment under the following conditions (lots, some, or no growth): LB plate (-) plasmid LB plate (+) plasmid LB+kan plate (-) plasmid LB+kan plate (+) plasmidExplanation / Answer
a) In transformation experiment, a control should be used to determine if contamination had occurred. This can be done by spreading non-transformed competent cells on control agar plate containg the antibiotic Kanamycin. If there is no contamination and the culture is pure, the untransformed cells without the plasmid should die on Kan+ plate. If however, there is any growth in the control plate, this can be the indication of contamination.
b) Since Kanamycin is bactericidal i.e. it kills the bacteria, transformed cells can not be immediately put on Kan containing agar plates. Instead, cells are first recovered/ revived at 37C for about half an hour in Kan free LB broth to let the cells express the antibiotic resistance gene. After that the culture can be spread on Kan+ plate. If the revival step is not done following results can be observed:
c) Heat shock is required for uptake of foreign DNA by competent cells. Competent cells spread without heat shock will show following result:
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