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Gel Electrophoresis Lab Report

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Results of Gel Electrophoresis

For the gel electrophoresis of the genomic DNA, a band of genomic DNA (roughly 23kbp) is observed near the top of the gel. Further down the gel is another block of bands (ranging from 100bp ~ 2000bp) which is the degraded RNA. My group’s sample (lanes 16&17) shows similar band patterns to that of other groups where genomic DNA and RNA bands are seen but in differing quantities of DNA since some bands appear more faint than the others.

In the gel electrophoresis of the plasmid DNA, 4 distinct bands of DNA are observed. The top band of roughly 23kbp is the nicked/relaxed circular plasmid. Below it is another band of roughly 9500bp of linear plasmid DNA. The following band of around …show more content…
Possible causes of the smudges are nuclease contamination which could have degraded the DNA or excessive amounts of DNA marker being loaded (Dube, n.d) . Hence, the sizes of the bands observed are largely estimated in this experiment.

Gel electrophoresis of the genomic DNA
A block of slightly smudged band of genomic DNA is observed. The bulky DNA molecule does not migrate far due to its large size (around 23kbp) which interferes in its mobility. The smear could be due to contamination of nucleases (Dube, n.d) which were not completely removed by EDTA in buffer PD1 used. EDTA chelates Mg++ which is required cofactor for many nucleases (Khosravinia & Ramesha, 2007). Hence, degradation of the DNA results in the diffusion of the DNA band.

RNA bands are also observed nearer the bottom of the gel. Since RNA molecules are much smaller (100bp~2000bp), they can move through the pores easily and reach towards the bottom of the gel. Contamination of RNA could be due to incomplete removal of RNA caused by insufficient RNase added. Additionally, the band of genomic DNA from the diluted sample in lane 17 is slightly fainter than of the corresponding band from the “neat” sample in lane 16 since there is lesser quantity of …show more content…
The first DNA band near the top of the gel is the nicked circular plasmid. The nicked circular has one DNA strand nicked by cellular topoisomerase during replication (Tirabassi, 2014). Hence, the DNA superhelical tension is relaxed allowing access for DNA polymerase. The resulting plasmid is a large and loose circle with slowest mobility in the gel. The following band, the linear plasmid is formed when both strands of DNA helix is cut at one site by restriction enzyme and is often due to contamination of endonuclease or harsh treatment in purification (Dellis, 2009). It moves faster than the floppy nicked plasmid due to its linear shape. The supercoiled plasmid migrate through the pores even faster due to its highly compact shape. Hence, its band lies further below the linear plasmid’s. The supercoiled conformation is formed when extra twists are introduced by DNA gyrase and is the native conformation in vivo (Dellis, 2009). Lastly, the circular single stranded plasmid moves the fastest due to its small size since it only contains a single strand of DNA of about 2500bp. The single stranded plasmid is produced when DNA is permanently denatured from excessively harsh alkaline lysis such as too long incubation as hydrogen bonds are unable to reform (Tirabassi, 2014). Diffusion of the last DNA band is also observed and could be due to contamination of nucleases which degrades DNA to smaller fragments

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