Josh Hunt
5/2/24
Biotechnology Lab
Plasmid Identification Project
In our class, we were assigned to incubate and identify our given set of DNA.
Using techniques taught in the B TECH 1015 curriculum, we’ve been able to get to the
point of successfully working with plasmids in order to get desired results. To do this, I
needed to created a proper plasmid. A plasmid is a small section of DNA that has been
removed from its chromosomal DNA within the cell. Within this plasmid, restriction
enzymes are added in order to cut the DNA at certain spots the enzymes recognize. I
then ran these substances through gel electrophoresis. This enables those studying
DNA to measure the basepairs of the sample they’re studying. The measurements of
these base pairs are able to predicted at sources like on the the website NEBCutter.
These predictions enable people studying DNA like me, to match our data with
correlating predictions from the website. In this way, I am able to predict the plasmid I
have been given from 3 possible plasmids. After thorough experimentation, I’ve
concluded that the plasmid I have been given is pKAN.
Having been given an enzyme with the code name 6025C19, I first decided upon
the recipe to use for the digest. Having been given a concentration from my teacher, I
determined to use a specific amount of plasmid for my recipe. We obtained both our
marker DNA, our restriction enzymes, and our reaction buffer from NEB, a science
company that stands for New England Biolabs. The next step was to then determine
how much and what kind of restriction enzymes I would use. After conducting some
research, I found the enzymes BglI, PstI, and BamHI to use for my incubation. I decided
to use BglI for one trail, and PstI and BamHI for another. This was due to the idea that
each of these enzymes cut the plasmids at least 2 or more times. In my second trial, I
used a fourth enzyme, HindIII, which I felt would give more accurate data. I then
determined the buffer necessary in order to allow proper incubation, which I found at
NEB’s website. After finding this, I was able to determine the amount of dH2O to add.
After assembling these, I allowed my digests to sit for about an hour and a half at 37 oC.
This was perhaps, in my opinion the most important part of the whole procedure, as this
determined whether or not the enzymes incubated the DNA. In my first experiment, my
BglI digest did not incubate, and in my second experiment, I found my BglI, as well as
my HindIII digests, in not fully incubate either. One conclusion drawn from this is that
these restriction enzymes need more time, or perhaps, a higher temperature, to
incubate. Using a .8% concentration of agarose, I added my determined amount of
agarose. I then measured out the amount of x10 TAE in a graduated cylinder, filling
the rest of the amount with water. I then transferred the contents to the flask containing
the agarose. After microwaving this for about a minute and 20 seconds, I let my solution
cool, and then poured it into the gel tray. I then ran my gel, which was at about 140 volts
the first time, and 130 volts the second time. The time these ran were about 40 to 50
minutes each, by the time they reached the 4.5 mark. I used a x10 TAE buffer, using a
tenth of 280 mL, which was the total volume. After my enzymes were ready, I used a
UV image system to take the picture of my gel. I found that only my PstI+BamHI was
the only digest I ran that actually incubated. From this, I was able to determine my
fragments. To do this, I first matched the ladders together, having 10 ladders the first
time (with a 0.5 kbp), and the second time which did not have the 0.5 kbp marker. I ran
a line through each of the lines shown in the digests that incubated. I then created a line
of best fit on a graph using the ladders. From that, using the equation excel gave me, I
was able to find the fragment sizes of the digest sections. My data correlated very nicely
with my predictions. However, after checking my predictions again, I found I had put the
settings on linear instead of circular. After correcting my mistake, I found that my data
did
not
work
predictions.
with
the
However,
after
consulting my teacher, I was
advised to consider if one of
my
cuts
never
actually
happened.
Thus,
I
allowed
to
was
determine
my
having
the
plasmid.
Figure 1
After
concentration being told to
me, which was 250 µg/mL, I
found my total miniprep of
plasmid to be about 2 µL.
Figure 1 shows my first trial,
when I did not include HindIII.
F my second trial, in which
neither my HindIII nor my BglI
Figure 2
digests incubated. However, I was able to determine the plasmid by looking at the data
given me by my PstI + BamHI incubation. When I plugged my data into excel, I found
highly positive results. I was able to find that my measurements were very accurate. My
DNA
Figure 3
Figure 4
fit
nicely
along
the
exponential
line.
Trial One Travel Distance (mm) Base Pairs
Difference from Predictions
PstI+BamHI #1
36.5
3462.7
-191.696
PstI+BamHI #2
48.6
1045.14 -122.137
Trial
Travel Distance (mm) Base Pairs Difference from Predictions
BglI #1
30.1
4113.6
80.4019
PstI+BamHI #1 31.9
3386.84
-115.843
PstI+BamHI #2 43.7
946.957
-23.9567
Predictions
BglI
PstI+BamHI
HindIII
pAMP
3263, 1118, 158
4468, 71
4539
pKAN
3139, 794, 261
2870, 923, 401
4194
pBLU
2121, 1710, 1576
3902, 1316, 197, 22
5437
pKAN (without second cut)
According
to
my
trials,
3271, 923
I
am
anywhere from 200 to 1000 base
pairs off from what I’m supposed to
be at, according to me predictions.
However,
after
consulting
my
teacher, I was able to determine
that
in
my
pKAN
PstI+BamHI
solution did not cut at the second
cut, thus I got a good fit that is +200 basepairs off. From that data, I
can safely conclude that my plasmid is pKAN, as my results match up with the base
pairs. A possible explanation as to why things happened the way they did is within the
incubation process. According to my knowledge, I would hypothesize that PstI does not
take a long time to incubate. On the other hand, BamHI does, which would explain why
my results were the way they were.
References

Nucleotide Sequences of Plasmids
Nucleotide Sequences of pAMP, pKAN, & pBLU Plasmids. Internet:
http://www.dnalc.org/resources/plasmids.html
NEBcutter V2.0
o NEBcutter V2.0. Internet: http://tools.neb.com/NEBcutter2/
Buffer Finder
o Double Digest Finder. Internet: https://www.neb.com/tools-andresources/interactive-tools/double-digest-finder
Mr. Scott
o Scott, R. (2014, May 2).
o
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