analysis the gel below Electrophoresis of circular DNA a) explain why lane 2 and
ID: 3167250 • Letter: A
Question
analysis the gel below
Electrophoresis of circular DNA
a) explain why lane 2 and lane 10 of Figure 5 show two bands (one band is darker and one is fainter)
b) why does it not make any sense to give a "size" for these molecules?
Figure 5. Agarose gel electrophoresis of plasmid DNA before and after digestion 1 2 3 4 5 6 7 8 9 10 11 reactions. Electrophoresis image of 1% agarose gel stained with GelRed and visualised under UV light. Sample preparation and gel electrophoresis was performed by Garry Penney Lane 1 Markers (SPP1 EcoRI digest) Lane 2 pK184hlyA undigested Lane 3 pK184hlyA digested with EcoRI Lane 4 pK184hlyA digested with Pstl Lane 5 pK184hlyA Lane 6 Lane 7 digested with EcoRI and Pstl Markers (SPP1 EcoRI digest) pBluescript digested with EcoRI and Pstl Lane 8 pBluescript digested with EcORI Lane 9 pBluescript digested with Pstl Lane 10 pBluescript undigested Lane 11 Markers (SPP1 EcoRI digest) Copy of Figure 2. SPP1 EcoRI size markers after electrophoresis Wells through 1% agarose gel When assigning sizes of marker bands on 1% agarose gels like this, determine the bands from the top (i.e. largest bands); the smallest fragments of DNA in the marker lanes have already run off the gel in Figure 5 SPP1 EcoRI size markers consist of the DNA of SPP1 phage digested with EcoRI restriction enzyme. Size (kb) markers are used in gel electrophoresis to estimate the sizes of DNA fragments of unknown length. 851 7.35 6.11 4.84 3.59 281 1.95 1.86 1.51 1.39 1.16 0.98 0.72 0.48 0.36Explanation / Answer
a. Lane 2 and lane 10 contain undigested plasmid samples.
They show more than one band because of the fact that plasmid DNA exists in three different form. They are
i. Supercoiled DNA
ii. Nikced DNA
iii. Linear DNA
Each form migrates at a different speed. Supercoiled DNA migrates faster as it is condensed. Linear DNA migrates slower.
b. We can not assign sizes to these bands of undigested DNA. This is because their migration is not directly correlated with their molecular size. The marker that is loaded on the gel is used to compare the sizes of linera DNA molecules only.
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