Transformation of Escheria Coli

1
GFPimps

• Transformation of Escheria Coli
• Boiling DNA Miniprep
• Analysis of plasmid DNA by Restriction Enzyme
  Digestion and analysis of plasmids by PCR

Group Members David Miller, Allyson Ford,
Brandon Sykes and Kristoff Fajardo
2
Summary

• The overview of our experiment contains several
  steps involving using bacteria as a factory to
  grow up our GFP plasmid. Using the library
  containing different genes, we were able to
  isolate a plasmid containing GFP. Our last
  experiment involving PCR and restriction enzymes
  verified if we had the GFP plasmid. Our series
  of steps were thought to be followed correctly,
  but, as seen in the final result, there was some
  sources of error. I believe the results of our
  experiment gives me a kind of driving force to
  continue this experiment successfully.

3
Introduction
Our experiments included transformation of
E. Coli, boiling DNA mini prep, analysis of
plasmid DNA by restriction enzyme digestion and
polymerase chain reaction (PCR). We expect in our
experiment to first isolate the glowing plasmids
that contain GFP from a jellyfish library in some
E. Coli. After more bacteria is grown we will
remove the plasmids from the bacteria. We will
conclude by conducting an experiment involving
PCR and restriction enzymes. This experiment will
reassure us that the plasmids selected contain
GFP.
4
MeThOdS

• Transformation of Escheria Coli
• Label tubes for plasmid DNA Library and place on
  ice.
• Pipet H2O and library into tube.
• Remove competent cells from dry ice, thaw and
  place on ice.
• Pipet cells into each tube and mix.
• Keep on ice.
• Place in water.
• Place on ice.
• Add LB at RT and place in water bath.
• Pipet on each plate Ara, AMP, AMP/Ara and spread
  from both tubes.
• Incubate plates upside down.
• Record of bacterial colonies for both plates.
• Decide plates you want to take a picture of under
  UV to look for bacteria that contain GPF
• Mark glowing and non-glowing colonies with
  circles.

5
MeThOdS

• Boiling DNA Miniprep
• Day 1
• Label snap cap culture tubes and pipet LB in
  each. Add ampicillin to each.
• Pick a bacterial colony that glows and doesnt
  glow and drop it into culture tubes.
• Grow Minicultures in a shaker overnight.
• Day 2
• Pipet grown bacterial culture into Eppendorf
  tubes.
• Spin at RT in microfuge.
• Pipet the supernatant off.
• Resuspend pellet in STET.
• Add Lysozyme.
• Mix.
• Sit at RT.
• Place in heat block.
• Centrifuge at RT.
• Pull out pellet and discard.
• Add Ammonium Acetate and ispropanol and mix.
• Place tubes in centrifuge with lids pointing up.
  Centrifuge at RT.
• Pipet off supernatant w/o disturbing smear on
  side of tube.

6
MeThOdS

• Analysis of plasmid DNA by Restriction Enzyme
  Digestion
• Label Eppendorff tubes (1P, 2P, 3P,4P,1H, 2H, 3H
  and 4H)
• Pipet plasmid DNA in tubes.
• Prepare mastermixes for P and H on ice.
• On ice distribute Mastermix P into tubes 1P 4P
  and Mastermix H into tubes 1H 4H.
• Transfer the reaction tubes in waterbath.
• Add 10x DNA loading buffer.
• Put H2O, plasmid prep 1 and 10x loading buffer.
• Load samples onto ararose gel with marker.
• Analysis of plasmids by PCR
• Make a dilution of plasmid DNA preps.
• Label the PCR tubes (1G, 2G, 0G,LG,1A, 2A, 0A and
  LA) and place on ice.
• Pipet DNA in tubes 1, 2 and L. Pipet H2O into
  tube 0.
• Assemble mastermixes (G and A) on ice
• Once distribute Mastermix G to PCR G tube and do
  the same for A.
• Keep PCR tubes on ice until ready for PCR
  machine. Then cap the tubes.
• Start the PCR program
• Add 10x DNA loading buffer and load onto agarose
  gel with marker.

7
Results
8
Figure 1
Figure 2
Figure 3
9
Conclusion
The conclusion of our experiments really had
their differences. The first experiment did not
come out correctly because the plates with
ampicillin did not allow bacteria to grow. We
did not have any bacteria that contained
plasmid.



For better results we could
have labeled the plates more accurately.. Our
second experiment had a good result because we
successfully isolated the DNA and plasmids from
the bacteria. The group really felt that all of
the procedures were performed correctly. This
led us into the third experiment. The third
experiment was dealing with PCR and the
restriction enzymes. Unfortunately, our
experiment did not go well because none of our
groups genes contained the GFP gene. Our
experiment did not glow.