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Caitlin Davis
Btech 1015
Plasmid Identification
Introduction
Plasmids are small circular pieces of DNA found in bacteria. They are commonly
used in biotechnology as vectors: DNA that can accept, carry, and replicate other pieces
of DNA (Thieman and Palladino, 2009). Restriction enzymes are enzymes that recognize
segments of DNA, called restriction sites, and cut them. These restriction enzymes are
used to cut open vectors so scientists can insert the DNA they want to be copied.
Gel electrophoresis is a method of separating segments of DNA, RNA, and
proteins based on size. This was accomplished by making an agarose gel with little
grooves or “wells” at one end, which was then loaded into a gel box. DNA samples were
loaded into the wells and exposed to an electric current created between two electrodes,
one on each side of the gel box. DNA is negatively charged, so it travels to the positive
electrode. Smaller pieces of DNA travel faster through agarose gel than the larger pieces,
thus separating the fragments of DNA by size.
The goal of this experiment was to identify which of four plasmids (pAMP,
pBLU, pGEM, and pGLO) the researcher was given. This is an important technique to
determine that the correct plasmid has been purified from a solution. It is accomplished
by digesting the DNA sample with restriction enzymes. If the restriction enzyme matches
the correct plasmid, then the enzymes are able to cut the plasmid DNA allowing it to be
replicated. To achieve the goal of this experiment, the unknown plasmid was cut into
fragments using restriction enzymes and separated using gel electrophoresis. Based on
the number of fragments and their location on the gel, it was determined which plasmid
was present.
Methods
The code name for this plasmid was Germanium (or Ge). The concentration of a
plasmid can be determined using spectrophotometry. The spectrophotometer was set for a
wavelength of 260nm. Next the absorbance of 50 L of water was measured for the
blank, and then the absorbance of a 5x dilution of the DNA sample was measured.
Finally the concentration was determined using the Beer-Lambert equation: A=Cl.
The DNA ladder used in this experiment was the 1kb DNA ladder from New
England Biolabs. Buffers and enzymes used were NEBuffer 4, EcoR1, and BamH1 made
by New England BioLabs. Refer to Table 1 for digest recipes. The digests were incubated
for 24 hours at 37 degrees Celsius.
Table 1 – Digest recipes
Tube
Plasmid
10x NEBuffer
BamH1
EcoR1
dH2O
Total
B
11.5 L
1.5
4 L
1 L
--
1 L
15
L
volume
E
11.5 L
1.5 L
--
1 L
1 L
15 L
E+B
11.5 L
1.5 L
1 L
1 L
--
15 L
Next a 1% agarose gel was created by combining 0.50 g of agarose and 50 mL of
TAE. The gel was heated in a microwave for 15 seconds following which the gel was
mixed by swirling it. This process was repeated until the agarose was completely
dissolved. The dissolved gel was poured into the gel tray, where it was allowed to
solidify.
7L of “6x loading dye + Sybr green” was then added to the plasmid samples and
4L was added to the ladder. The agarose gel tray was then placed in the gel box, which
was filled with TAE buffer until the wells were submerged.
After loading the gel with the ladder and digests, the gel was run at 150 volts for
40 minutes. An image of the gel was observed and recorded using a UV Transilluminator.
An initial determination of the size of the plasmid fragments was made by comparing the
digest bands to the ladder. After this initial determination was made, a more accurate
determination was accomplished by using a standard graph. The plasmid fragment sizes
were compared to the expected fragment sizes which were found using the website
http://tools.neb.com/NEBcutter2/index.php. From this comparison, the actual plasmid
present was determined.
Results and Conclusions
The starting concentration of the plasmid
was 17.4 ng/L. Figure 1 shows a UV image of
the finished gel. See Figure 2 for ladder band
sizes. Lane B is the BamH1 digest, lane E is the EcoR1 digest, and lane E+B is the
double digest EcoR1 + BamH1. Figure 3 shows the standard curve graph used to
determine the sizes of the fragments created by the digests. The R2 value is a measure of
how closely data fits a given curve. The closer the R2 value is to one, the better the fit.
The R2 value of this experiment’s data set as compared to the standard curve graph is
0.9959, which indicates a very high level of agreement. Table 2 shows the approximate
fragment sizes for the digests based on the standard curve. Tables 3 through 6 and
Figures 4 through 7 account for all the digests because the same two enzymes are being
used (all made on NEBcutter). Table 3 shows the expected fragment sizes for pAMP
after being digested by the enzymes. Table 4 shows expected fragment sizes for pBLU
after digestion. Table 5 shows expected fragment sizes for pGEM after digestion. Table 6
shows expected fragment sizes for pGLO after digestion.
Figure 4 shows a restriction digest map for pAMP after being digested by BamH1
and EcoR1. Figure 5 shows a restriction map for pBLU. Figure 6 shows a restricion map
for pGEM. Figure 7 shows a restriction map for pGLO.
Based on these results, the experiment was inconclusive as to the specific plasmid
that was present. Table 2 shows that the fragment sizes range from 5.1 kb to 6.2 kb. Lane
B’s size of 6.2 kb is far larger than the size of the largest plasmid pBLU, which is 5.4 kb.
Lane E has a total size of 11 kb, while Lane E+B has a total size of 11.3 kb. These values
render any comparison of fragment sizes invalid.
However, a comparison of fragment quantities indicates that the plasmid present
is pGEM. Referring to Figure 6 it can be seen that there is only one place where BamH1
digests pGEM. Lane E in Figure 1 had two digests. Figure 6 shows that there are two
places where EcoR1 digests pGEM. It cannot be pAMP because that would mean there
was only one band or digest in lane E. The same argument goes for pBLU along with the
fact that the two bands in the double digest would have been placed much further apart
because of the size difference. If the plasmid was pGLO, then there would have been
three bands in lane B and four bands in the double digest (E+B). This leaves the
unanswered question that if the plasmid is indeed pGEM, why are there only two bands
in the double digest lane? Since the digests are close to the same size, I believe there are
three bands, but the single bright band on top has covered one of them up.
References
 Thieman, William J. and Palladino, Michael A., 2009: Introduction to
Biotechnology. Pearson Education, Inc., San Francisco.