RESULTS

Figure 1. Analysis of plasmid DNA by agarose gel electrophoresis after restriction

enzyme digestion. Treatments of 4 μL purified E. coli plasmid DNA are labeled. 1%
agarose gel run at 100V for 35 minutes.

Table 1. Approximate sizes of digested plasmid DNA samples obtained from gel
image.
Lane Treatment Size (bp)
6 Plasmid DNA 1 4000
5 Plasmid DNA + 1 μL EcoR1 1 N/D
4 Plasmid DNA + 1 μL HindIII 1 5000
3 Plasmid DNA 2 5000
2 Plasmid DNA + 1 μL EcoR1 2 6000
1 Plasmid DNA + 1 μL HindIII 2 6000
Note: Lane 5 lacks a defined band so it is listed as N/D for No Data. Lane 4’s
measurement is of the most defined band.
DISCUSSION
In exercise 7, E. coli plasmid samples were purified using alkaline lysis
method. In exercise 8, purified samples were digested with restriction enzymes
EcoR1 or HindIII, and run through agarose gel at 100V for 35 minutes.
The difference in size between plasmid samples treated with EcoR1 (lane 2)
and untreated plasmid in lane 3 and 6 was expected. According to information
presented in the lab, EcoR1 generally has 1 restriction site on an E. coli plasmid
which makes the plasmid linear after being cut. Linear DNA travels through agarose
gel slower than supercoiled DNA in samples of untreated plasmids, explaining the
size difference of around 1-2 kbp between the plasmids of 2 treatments.
Lane 5 was not expected but could be explained by mistakes during the
plasmid preparation. The lack of bands in this lane might indicate that the sample
had too little plasmid DNA to visualize. During the lab, my group faced difficulties in
transferring plasmid DNA into a new microfuge and noticed that a part of the
sample was stuck on the tube’s wall after mixing.
Aside from the observations discussed above, there were also some
unexpected results. Since HindIII can have 2 restriction sites on an E. coli plasmid,
digested samples were expected to produce 2 separate bands. However, there is
only 1 band for both samples in lane 1 and 4. HindIII-treated plasmid in lane 1 ran
slower compared to untreated plasmid DNA (lane 3) but at similar pace with
EcoR1-treated plasmid in lane 2, which suggested that the plasmid was cleaved at
one site and linearized. The sample in lane 4 might have part of it nicked or
linearized as extra bands of larger size were seen.
There are hardly-visible bands of smaller size under plasmid samples in lane
1 and 2. These bands are too large to be considered a product of restriction
enzymes, I suspect that these could be plasmid DNA in a different structure. Sayers
et al. (1996) suggested that these ghost bands can be cyclic coil DNA that often
arise in alkaline conditions, which is possible since we used the alkaline lysis
method for plasmid purification.
Almost all lanes have blurry bands of low molecular weight at the bottom of
the gel. Due to the size, I believe that they could be small nucleic acids. However,
their appearance is very similar with the bands in gel electrophoresis images in an
article by Frerix et al. (2006). The plasmid samples in this study were also treated
with the alkaline lysis method and the authors determined that these bands are
RNA. This might indicate incomplete degradation of RNA by RNAse during the
purification process.

CONCLUSION
When cleaved by restriction enzymes, plasmid DNA is linearized and travels slower
in agarose gel than supercoiled circular plasmid DNA of similar size.
REFERENCES

Frerix, A., Geilenkirchen, P., Müller, M., Kula, M.-R. and Hubbuch, J. (2007),
Separation of genomic DNA, RNA, and open circular plasmid DNA from
supercoiled plasmid DNA by combining denaturation, selective renaturation
and aqueous two-phase extraction. Biotechnol. Bioeng., 96: 57-66.
https://doi-org.uml.idm.oclc.org/10.1002/bit.21166

Sayers, Evans, D., & Thomson, J. B. (1996). Identification and Eradication of a

Denatured DNA Isolated during Alkaline Lysis-Based Plasmid Purification
Procedures. Analytical Biochemistry, 241(2), 186–189.
https://doi.org/10.1006/abio.1996.0397