In the left lane 5 bands were obtained by digesting lambda DNA with EcoRI. This depicts that the DNA has been digested as first few lanes are visible instead of 12 visible bands, this is due to weak staining of DNA, hence only larger bands are visible. Also, agarose gel does not have a high resolving power as compared to polyacrylamide gel (PAGE). They are still used for electrophoresis because their construction is easy and quick to run.
In the middle lane a single band was obtained by running pUC19 (‘p’ denotes plasmid and ‘UC’ denotes University of California) without digesting it with EcoRI, thus being a circular molecule it was able to move through the gel more quickly than linear molecules and ended up further down the gel. Hence, this a control experiment where restriction endonuclease was not used. The molecular weight of pUC19 is 1.75*106 Daltons.
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In the right lane only a single prominent band was obtained located above the undigested pUC19 DNA of the middle lane. This single band is of pUC19 DNA which has been digested by EcoRI indicating that digestion has occurred properly. pUC19 has one EcoRI restriction site which means after restriction digestion a single long linear DNA molecule is obtained which travels slowly through the pores of agarose gel as compared to circular pUC19 plasmid DNA which can also form coils and supercoils. These travel more quickly through the gel and end up further down the lane. Another reason for this observation is, may be one long linear DNA molecule has joined with another such molecule by their sticky end which have been made by EcoRI, to form longer DNA molecules that travel slowly due to their size/weight or more than one circular fragment has joined to another to generate larger/heavier molecules. To obtain higher resolution result ligation of plasmids could be prevented by performing phosphatase treatment which causes dephosphorylation of the 5’end thus preventing formation of phosphodiester bonds between the 3’OH and 5’P residues. To enhance resolution, electrophoresis can be carried out for longer that will allow the bands to separate and resolve more clearly.
An improvement of this experiment can be done by using polyacrylamide gel instead of agarose gel because of it’s high resolving power though, they are difficult to construct and takes longer to run. Hence, higher resolution results can be obtained. Lab-on-a-chip is a fully automated, high throughput system which provides reproducible data. It reduces time consumption, sample and reagent use. This leads to reduction in the amount of hazardous waste produced and per-sample cost. Thus, it is advantageous over agarose gel electrophoresis. Percentage of gel used for electrophoresis could also be taken into consideration. Higher the percentage of agarose, the smaller the pores formed in the gel which makes it difficult for the linear DNA molecule to travel through the gel. Hence, by reducing the percentage of the gel from 0.8% to 0.6% larger DNA molecules will be able to resolve in the gel.
Also, smaller fragments of DNA stain lighter than larger fragments so, in order to elevate resolution of bands staining could be done for longer which will allow the smaller DNA fragments to take up the stain properly and thus will be visible to us. Alternative dyes that can be used instead of Bromophenol blue are SYBR Gold and SYBR Green because they are highly sensitive dyes and safer than some other dyes like Ethidium Bromide. Other dyes which could be used are Eva Green and Acridine Orange. A table (table 1.b) has been given that depicts the different stains that can be used for electrophoresis.
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The Analysis And Characterization Of DNA By Restriction Endonuclease Digestion.
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