Analyzing DNA: Tracking GFP in Bacteria Cells

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Analyzing DNATracking GFP in Bacteria Cells
Presented By Andy Wells, Brian Madrid, Joseph
Attia, Jamiah Harris
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Introduction

• Glowing jellyfish contain a gene called GFP,
  which codes for proteins which make the jellyfish
  glow. Were using plasmids to extract that DNA and

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Summary

• We transformed the GFP gene found in a genetic
  library of a glowing jellyfish into the bacteria
  E-coli.
• We next extracted plasmids with the GFP gene from
  the E-coli.
• We last cut the GFP gene out of the plasmid and
  reproduced them in two different ways.
• PCR- We copied the GFP DNA with primers, and then
  heat shocked them repeatedly to promote
  exponential growth of the GFP DNA.
• RE- We used restriction enzymes to cut at
  promoter sites, to isolate and size the GFP DNA.

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Materials Used

• 1 aliquot of competent E. Coli cells.
• Plasmid DNA library.
• Luria Broth
• Ice bucket
• 20, 200, 1000 p pipetmin.

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Procedure I

• We used competent Escherchia coli cells in which
  to implant our plasmids which contained either
  the GFP or Ampicillin gene.
• We put 2.5 microliters of water into two
  Eppendorf tubes. One mard O for water and one
  marked L for library.
• Next, we put 25 microliters of cells into each
  tube. We also added Luria Broth to the tubes of
  both water and the library solutions.

• In order to get the plasmids into the bacteria,
  we had to perform a couple of heat shocks. One
  was at 42 degrees Celsius for 30 sec. The other
  was at 37 degrees Celsius for 15 min.
• We used 8 petri dishes to grow the bacteria.
• We placed the solution with the O on 4 dishes
  one with no marks, one with 1 mark, one with 2
  marks, and one with 3 marks.
• We did the same for the library solution.

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Procedure I (cont)
After the heat shocks, we then spread the
solution from the Eppendorf tubes on to a
prepared petri dish, whish either contained Luria
broth and 1.5 agar or Luria broth, 1.5 agar,
and 1 arabinose, or luria broth, 1.5 agar, 1
arabinose and 100 ug/mL ampicillin.
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Conclusion

• The purpose of the entire procedure initiating
  on July 17, 2001, and commencing on July 21, 2001
  was to manifest numerous concepts. Specifically,
  1) the capability of creating multiples of a
  particular genethe GFP or the glowing gene, 2)
  the process of extracting DNA from a cell, 3) the
  ability to dissimulate or distinguish a certain
  type of DNA. The physical principle we verified
  is that despite the surrealist idea of this genre
  (Molecular Biology), we were able to scrutinize
  the matter at hand most efficiently. Though our
  results were quite satisfactory we encountered
  some errors. The first notable residual that
  occurred was during the pipeting rather than
  pressing the pipet button in the appropriate
  manner we pressed in excess, thus causing there
  to be an oversupply for every substance.
  Furthermore, in our first procedure (
  Transformation of E. Coli) we thawed the
  competent cells for to long, which possible may
  have resulted in some sort of variability in the
  state of the cells.

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The End