Transformation and Electrophoresis
How can a plasmid be engineered to include a foreign piece of DNA and how does gel electrophoresis separate DNA molecules present in a mixture?
If the pGLO plasmid is inserted into competent Escherichia coli cells, then the transformed bacteria will be resistant to ampicillin and will glow green under UV light. If samples of DNA are cut using certain restriction enxymes and separated using gel electrophoresis, then the smaller the DNA fragment cut, the greater the distance it will travel in the gel.
The control plates used in transformation are the LB and second LB/Amp plates marked …show more content…
To Analyze the Results:
12. Observe the plates, “+” and “-“ and count the colonies by looking through the bottom of the plates (use a permanent marker to mark each colony as it is counted). Do not open them up. If cell growth is too dense to count individual colonies, then record “lawn.”
13. After your observations and collection of data, reseal the Petri dishes by taping them together and give them to the instructor for dispersal. You will be observing plates that were prepared by the last section.
14. Transformation efficiency is expressed as the number of antibiotic-resistant colonies per microgram of pAMP. The transformation efficiency was determined by using this formula.
• The total mass was calculated by multiplying the volume by the concentration (10 μl x 0.005 = 0.05 μg).
• The fraction of the total cell suspension that was spread on the plate was calculated by dividing the volume of μl spread by the total volume (100/500 = 0.2 μl).
• The mass of the pGLO in the cell suspension was calculated by multiplying the total mass of the pGLO by the fraction spread (0.05 x 0.2 = 0.01 μl).
• The number of colonies per μg of plasmid, or Transformation Efficiency, was calculated by dividing the colonies observed by the mass of pGLO spread (1880/0.01 = 188,000).
• The transformation efficiency for the +LB/Amp plate is: 8.8 x 104.
• The transformation efficiency for the