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

• A glowing jellyfish contains a gene that
  causes it to glow. The task at hand was an
  attempt to transfer that specific gene, GFP,
  into competent E. coli bacteria cells which was
  cultured. Then we used PCR to amplify the amount
  of DNA that contained the GFP of ampicillin
  resistant gene. We used Restriction Enzyme
  Digestion to cut the strands of DNA.

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Summary

• The transfer of the GFP gene found in a genetic
  library of a glowing jellyfish into the bacteria
  E-coli.
• The extracting of the plasmids with the GFP gene
  from the E. coli.
• The cutting of the GFP gene out of the plasmid
  and reproduction synthesized by 2 methods.
• PCR- coping of the GFP DNA with primers, and then
  heat shocking them repeatedly to promote
  exponential growth of the GFP DNA.
• RE- restriction enzymes to cut at promoter sites,
  to isolate most of the GFP DNA.

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Materials Used

• Reagents
• one aliquot competent E. coli (Escherichia coli)
  cells in dry ice
• 1 ml H2O
• plasmid DNA library at 0.1 µg/µL (in central
  location)
• 2 LB plates of each
• no mark LB 1.5 agar
• 1 mark LB 1.5 agar 1 arabinose
• 2 marks LB 1.5 agar 100µg/ml Ampicillin
• 3 marks LB 1.5 agar 1 arabinose 100µg/ml
  Ampicillin
• 5mL of LB
• 37 and 42 degrees Celsius water baths
• ice bucket
• Sterile distilled H2O

• Plasmid DNA preps 1,2,3,4
• 10x buffer 2 (500mM Tris.Cl pH8.0, 100mM MgCl2,
  500mM NaCl)
• RNAse A (10mg/ml)
• Restriction enzymes PstI (10unit/µL) and HinDIII
  (10unit/µL)
• 10x DNA loading buffer
• Solutions
• STET
• 8 Sucrose
• 50mM Tris-Cl, pH8.0
• 50mM EDTA, pH8.0
• 5 Triton X-100
• filter sterlize
• TE
• 10mM Tris.Cl pH8.0
• 1mM EDTA, pH8.0
• 200mLs
• 16.0g
• 10mL of 1M stock

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Procedure I

• The competent Escherchia coli cells in which to
  implant our plasmids was used, which contained
  either the GFP or Ampicillin gene. Water was put
  into two Eppendorff tubes. One was marked O for
  water and one marked L for library. Next, some
  cells were put into each tube. Also, some Luria
  Broth was added to the tubes of both water and
  the library solutions. In order to get the
  plasmids into the bacteria, a couple of heat
  shocks had to be performed. One was at 42 degrees
  Celsius for 30 sec. The other was at 37 degrees
  Celsius for 15 min. 8 petri dishes were used to
  grow the bacteria, which were then placed in the
  solution with the O on 4 dishes one with no
  marks, one with 1 mark, etc. The same was done
  in the library solution. After the heat shocks,
  the contents of the tubes on various petri dishes
  were emptied which contained different solutions
  that catalyze the growth of bacteria.

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Procedure II
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Procedure III

• PCR
• First, after adding TE to our two tubes of DNA
  to stabilize the pH levels, make a dilution of 2
  plasmid DNA preps and a library, with water.
  Next, pipet the dilutions and water into the
  respected PCR tubes. Then mix the 2 mastermixes.
  Mastermix G and Mastermix A were both made with
  sterile, distilled H20, 10xPCR buffer, dNTPs,
  Taq enzymes. In the Mastermix G tube also add a
  GFP starting primer and a GFP ending primer. In
  the Mastermix A tube also add an AMP starting
  primer and an AMP ending primer. Then distribut
  each Mastermix into the corresponding tubes. Then
  place the PCR tubes into the PCR machine to
  rapidly reproduce the primer selected DNA. Then
  put a buffer into each PCR tube, to make the DNA
  show in the gel electrophoresis, and then place
  the DNA into the gel wells.
• Restriction Digestion
• Initially, label eight Eppendorff tubes 1P, 2P,
  1H, 2H Then pipet 5µl of plasmid DNA in the
  tubes. Then prepare Mastermixes (P and H) for
  each enzyme on ice. Distribute 20µl from
  Mastermix P to reaction tubes 1P4P, and do the
  same for Mastermix H. After transferring them
  into a 37 degrees Celsius water bath for 1-3 hrs,
  add 3µl of 10x DNA loading buffer. Label a fresh
  tube 1- and pipet 20µl H2O, and add 5µl of
  plasmid prep 1 and 3µl of 10x loading buffer.
  Then conduct the process of electrophoresis and
  examin the results.

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Results

• PCR Analysis
• The results for the Polymerase Chain Reaction
  were not conclusive. The PCR experiment was
  hampered by either human or scientific error. One
  thing that possibly could have gone wrong was the
  Lysozyme wasnt kept on ice long enough and it
  became bad. This particular group also had a
  slight problem with pipeting the correct amount
  of solution into the different tubes. This is one
  of the major factors that could have hindered the
  results for the PCR experiment. Another important
  factor that must be taken into account is the
  fact that all of the laboratory equipment we used
  in this experiment was new. This could mean that
  all of the materials used could have been
  defective, or even just one or two. There are too
  many variables in this set up that could have
  been off. Thus, one can not single out one factor
  over the other. In conclusion, in order to obtain
  the desired results from this lab, the lab team
  should go over this lab procedure and then repeat
  the experiment to find the spot(s) where the lab
  went wrong.
• Restriction Digestion
• The first Pst1 enzyme was hypothesized that it
  would cause the DNA to glow, which it did not.
  However, the second did, which was suspected. The
  third was thought to not glow, but it did. It was
  assumed that the fourth would glow, but it did
  not. Therefore, with the Pst1 enzyme, the
  hypothesis was 25 correct, or 75 wrong.
• With the HindIII, the results were much more
  accurate. It was assumed that the first DNA
  strand would glow, which it indeed did. It was
  also thought that the next two would be linear
  plasmids, due to their lack of the GFP gene,
  making them non-glowing DNA strands. Lastly, it
  was theorized that the last DNA strand would
  glow, expressing the GFP gene, and it did indeed.
  So in this case, the hypothesis was 100 correct.
  

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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 verified is
  that despite the surrealist idea of this genre
  (Molecular Biology), the lab group was able to
  scrutinize the matter at hand most efficiently.
  Though the results were quite satisfactory some
  errors were encountered.
• The first notable residual that occurred was
  during the pipeting rather than pressing the
  pipet button in the appropriate manner, it was
  pressed in excess, thus causing an oversupply of
  every substance. Furthermore, in the first
  procedure (Transformation of E. Coli) the
  competent cells were thawed for to long, which
  possible may have resulted in some sort of
  variability in the state of the cells. Secondly,
  in the aforementioned procedure the petri dishes
  were labeled incorrectly which not only caused
  some confusion among the lab group, but also
  inhibited the possibility of growing bacteria
  cells with the GFP gene. In addition, during the
  second procedure (Boiling DNA Miniprep), the two
  teams fondled the four Eppendorf tubes, thus
  creating a potential bias. Finally, it was
  believe that certain error may have been sprouted
  from the general atmosphere of the laboratory,
  specifically, it wasnt an isolated environment.
  It is apparent that in the field of Biology any
  slight variation in anything (eg. especially
  temperature, foreign organisms, etc.) can cause
  a great deal of variation in the final results.

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Conclusion (Cont.)

• A particular method of improving the procedure
  would be to carry-out the undertaking in a cold
  room. Furthermore, maybe allowing a maximum of
  two group members for each procedure, thus
  probably enabling the procedure to be more
  accurate.

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We think, therefore we are