Case Study Gel Electrophoresis

A0123942_Gel Electrophoresis Report

Name: Lee Zixuan

Process of Gel Electophoresis:

Gel electrophoresis in this case involves the placement of both genomic and plasmid DNA inside the wells of the agarose gel, together with a gel loading buffer. The agarose gel contains mini pores such that when an electric current is switched on, it would be able to separate the bigger segments of DNA bands from the smaller ones. As DNA is negatively charged due to the phosphate group, it would move towards the positive electrode. Smaller molecules of DNA would be able to move faster than the bigger molecules of DNA. Upon completion, the separated fragments of DNA can be visualized under UV light and the distinct bands could be seen.

Lambda HindIII-cleaved

Upon closer inspection, 5 distinct bands could be deduced from the photo. This is largely coherent with the other bands seen in the other lanes, with a clear reference done to lanes 1, 2, 12, 13, 14. The possible explanations as to why the other bands could not be seen distinctly in the other lanes is probably due to the genomic DNA being treated with too much phenol and chloroform, thus contaminating it. A pure sample should contain a ratio of around ~1.8 when compared to the absorbance ratio at 260nm and 280nm. Also, the concentration used for the Gel Loading buffer may be too little, such that when electrophoresis occurs, the gel is not able to fully submerge the DNA, leading to diffusion of DNA, which could be seen in lanes 7, 8, 9, 17, 18 where the bands exists as blurred smeared

The 3 bands moved 0.9cm, 1.1cm and 1.5cm from the agarose well respectively (measured from original photograph).When compared to the marker, we can assume that all the 3 bands of DNA are rather large in size compared to genomic DNA, at roughly around 5500-12000 base pairs. The photo showed us that the plasmid DNA is probably adopting a linearized form. When compared to supercoiled genomic DNA form, these linearized forms would contain a higher amount of base pairs and thus, the distance traversed would be at a minimum. Plasmid DNAs are usually in a supercoiled form but as seen in this case, they must have been digested with a restriction endonuclease enzyme that cuts both DNA strands of the plasmid at 2 different recognition sites (therefore giving 3 bands). This would result in a linearized form of DNA, which contains larger amounts of base pairs, resulting in slower traverse speed in the agarose