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Plasmid Identification Project Paper

James Mitchell Sanborn
May 2, 2014
Biotechnology 1015
Richard Scott
Salt Lake Community College










In this research paper I will be explaining the purpose behind my
experiment, my results, and my conclusion about my experiment. The purpose of
this experiment was to identify what kind of a plasmid I was given. My job was to
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identify this plasmid in the most accurate way as possible using all of the
techniques we were taught. A plasmid is a circular piece of DNA. Its purpose is to
carry information about who gave it that DNA. For Example, if it was found in an
animal it would be used to carry genes for that animal.
In order for us to identify what kind of DNA the plasmid is carrying we need
to cut the DNA into unequal segments. To do this we must use something called a
Restriction Enzyme. Its purpose is to find a specific segment of DNA and cut at a
certain point. This point will almost always be unequal from the other segments.
After using Restriction Enzymes to cut the DNA into unequal segments of
DNA we now need to measure the length of the DNA. To perform this we must
use a tool called an Agarose Gel. We then will submerge this gel into a tank
containing the DNA into a salt solution that conducts energy. DNA will always run
to Positive energy so we know which way it will go. We apply a certain amount of
voltage to the gel for a certain amount of time. As the DNA goes through this gel it
will separate the smaller fragments from the larger fragments, with the larger
fragments taking longer to run to the positive energy source. We then can measure
where cuts actually happen and compare it to our predictions on where it will cut.
This is a very important experiment due to the fact that in Biotechnology we
need to figure out what kind of DNA something is holding as well as what it
performs. All the information about everything that is alive is in these pieces of
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DNA. They all hold genes as well as information on what certain cells are to
preform.
I will now tell you how I performed the experiment but before I do that I
would like to give you a general strategy of what I did. First we use restriction
enzymes to cut the fragments. Next we run it in gel electrophoresis. Finally we
read it using a UV light. With that done we will now go onto the details on how I
conducted the experiment.
The plasmid that I was given had the code 4216E11. The first thing that I did
was I labeled three 1.5mL tubes: C, S, and D. These stood for Control, Single
Digest, and Double Digest. Next I added 1.5 microliters of DNA to each tube. The
following step was to add 2 microliters of 2.1 Buffer to tube S. I then added 2
microliters of 3.1 Buffer to tube D. The next step is to add your amounts of dH
2
O.
For Tube C I added 16.5 microliters. For Tube S I added 15.5 microliters. For
Tube D I added 14.5 microliters. The final thing I added to the tubes was add my
restriction enzymes. For Tube S I added 1 microliter of Bg1I. I then added 1
microliter each of BamHI and NdeI. I then vortexed and centrifuged the tubes. *All
Restriction Enzymes were bought from New England BioLabs.
In order for the restriction enzymes to start working we need them to be in
an area that is in the suitable conditions. According to the company that produced
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the enzymes it told me these restriction enzymes were in suitable conditions at
37
o
C. So I then proceeded to digest these tubes in a 37
o
C heat block.
While I was waiting I produced an Agarose Gel. I first acquired the tank and
gel holder with the part that will mold the gel into its right shape. Then I went over
to the weighing station and got 0.48g of Agarose Powder. I then diluted that with
5mL of 10x TAE and 45mL of dH
2
O. I heated that up for 30 seconds and then
heated it in 15 second increments twice. I let it cool off for a little bit and then
added 1 microliter of Ethidium Bromide. I gently swirled and poured into the
mold. Following this I placed a 10 well comb on the side of the gel making sure it
fit into the respective notches. I then waited for 30 minutes for it to finish.
When the digest had finished after the hour I removed all three tubes from
the heat block and centrifuged them all. I then made 250mL of the salt solution for
the gel electrophoresis. I took 25mL of the 10x TAE and added it to a graduated
cylinder. I then brought to a volume of 250mL using dH
2
O. I then poured it all into
the tank. I then loaded 5 microliters of 6kb ladder from New England Biolabs to
the first slot, 20 microliters of Tube C to the third slot, 20 microliters of tube S in
slot 5, 20 microliters of tube D in slot 7, and then loaded 5 microliters of 6kb
ladder to slot 9. I let the gel run at 140 Volts for 50 minutes. When the gel was
done working I went over to the UV light and took a picture of my gel in the light.
Results and Conclusion
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The original concentration of the plasmid was 250ng/microliter.
Single Digest Predictions

3408-2131 3263
2290-3407 1118
2132-2289 158

In conclusion to this lab I believe that my plasmid would have to be pAMP
based off the evidence that I have here. My cut was in the 4,000 area and this is the
closest of all three of the choices. Also the reason I am not putting my Double
Digest down is because it never did digest. I believe that this could have been
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closer but from the evidence that I have here it shows that the digest is not
complete and would need to be completed in order for a closer comparison.
Link to my graph:
https://docs.google.com/spreadsheets/d/1H9xrI1qMwR2Sk3RnsdUYdJWgZqPAp
CQiunQGenz3kd0/edit?usp=sharing