Name: Dilay Rıdvan 12/31/2018

ID number: 54014
Section: 2

EXPERIMENT 7 – PLASMID DNA ISOLATION

 Ladder name: GeneRuler 1kb DNA ladder (Thermo Scientific)
Aim
What is the aim of plasmid isolation experiment?
The main goal of this experiment is to isolate pasmid DNA (geneX in pCMV-Sport6) from
overnight grown E. Coli culture by following the kit protocol and to digest plasmid DNA
using restriction enzymes HIND III & XbaI.
 Why we need to isolate plasmid DNA, give one example for its applications.
Plasmids are important tools for recombinant DNA technology and they have features like
antibiotic resistance. Plasmids can replicate independently from genomic DNA therefore one
czn produce the product of gene of interest as a tool. However plasmids should be isolated
from the bacteria and other celluler components in order to carry the gene of interest.

Results

Figure 1
Group 2 datas will be used

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  How many bands did you observe in your ‘uncut’ sample? How many bands were
you expecting in which size? Explain briefly.
The first column of. Group 2 data at figure 1 represents the “uncut” sample. 4 bands are
observed at this column. However, plasmid DNA may appear in one of the five
conformations, which run at different speeds in a gel during electrophoresis: Super Coiled
DNA, Nicked Open Circular DNA, Relaxed Circular DNA and Linear DNA. According to
their electrophoretic mobility, different plasmid conformations can be observed in relative
positioned bands.

 How many bands did you observe in your ‘restriction cut’ sample? How many bands
were you expecting in which size? Explain these bands briefly.
The second column of Group 2 data at figure 1 represents the “restriction cut” sample. 1 band
is observed at this column however 2 linear bands were expected to be seen. Because with the
restriction enzymes Hind III and XbaI, plasmid DNA was cut into two segments: one
representing the gene of interest and the other one is plasmid backbone.

Discussion
 Was your plasmid isolation experiment successful? How do you know it was
successful or not? Briefly explain.
In the figure above, the plasmid DNA are seen with agarose gel electrophoresis. In a
successful experiment the marks of cut and uncut samples must be observed clearly at
different columns. According to our results, first column has 4 bands with different lengths.
These bands are representing the supercoiled DNA’s and second column is representing the
chromosomal DNA.

 Was your restriction enzyme digestion experiment successful? Briefly explain.

Our restriction enzyme digestion experiment was nearly successful since the appearance of
the different sized bands alone in “cut” column shows that the cleavage of plasmid DNA are
occurred. 2 linear lines were expected to be seen on the column, one showing the geneA and
the other showing the plasmid backbone. According to our results these lines had been able to
be visualized as an expected outcome.
The Restriction enzymes recognize the place where they connect on DNA so they can cut the
section desired. Cleavage of DNA into fragments or near specific recognition sites made it
possible to observe these two different alignments of proteins regarding the effect of isolation.
Also if isolation is successful we should obtain plasmid soluble supernatant and pellet
samples as our qualitative observations.

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  Explain why we used RNase in Solution I. In the Figure.1. below, determine whether
there is RNA contamination or not?
The most important point of the plasmid DNA isolation was to obtain the desired plasmid
DNA without other cellular components such as RNA, lipids, proteins and genomic DNA
(gDNA). So the removal of the undesired cellular component, RNA, was aimed to be
achieved by the addition of RNase in the solution. When looking at the figure given, it can be
stated that there is RNA contamination. In Figure 2 the small RNAs can be seen at bottom (in
lower bands) since their molecular weight is much smaller compared to DNA. Therefore, the
blurred and pale thick bands can be said that they correspond to RNA contamination.1

Figure 2 Agarose gel electrophoresis

 Please use below Figure 2. to answer questions



Figure.2: A plasmid is 8 kb long. The marks on the plasmid indicate restriction site positions
for the enzymes EcoR1 and BamH1.

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  What types of DNA fragments would be result if you cut a plasmid shown in the
figure with the restriction enzymes both EcoR1 and HindIII?
Since the length of the plasmid is 8 kb if we cut the plasmids with both restriction
enzymes, we’d end up with four fragments per plasmid: Two 2 kb in length, one 3 kb, and
one 1 kb.
 Please draw the agarose gel image of the uncut plasmid DNA with size and the
restriction digestion products with size (You can use the given image below).

References (5p)

1 https://www.mybiosource.com/learn/testing-procedures/plasmid-isolation