Electrophoresis of very small molecules and very small size differences a) Cutti

Question

Electrophoresis of very small molecules and very small size differences

a) Cutting of pBluescript with both EcoRI and PstI should produce and 11 bp piece of DNA and a piece just under 3.0 kb. Why then is there only one band on Lane 7?

b) Do you expect the molecules represented by the bands in Lanes 7, 8 and 9 to be exactly the same size? Explain.

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 0.36

Explanation / Answer

A. Both EcoRI and Pst1 both restriction enzymes located near to each other 11bp distance. So even if we double digest the plasmid it shows the same band. The 11bp band is not enough to visualize in the gel.

B. Yes, Lanes 7, 8 and 9 to be exactly the same size.
Consider 6Kb plasimd. EcoRI located at 3Kb region, so digest and provides 6Kb linear plasmid. PstI located at 3.01Kb region so, it also provide same 6Kb linear plasmid. lane 10 undigested so it is circular so it move further in the gel even it is 6kb plasmid.

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