Pb322 Lab Report

Discussions: In the experiment, the plasmid pBR322 would be cut off by restriction enzymes EcoR1 and Pst1 at the respective restriction sites into smaller fragments. The reaction mixture which contained plasmid DNA, enzymes, buffer for enzyme and distilled water was centrifuged, incubated and frozen. In the next session of practical class, preparation of 0.8% agarose gel, gel casting, gel staining and running the gel would be carried out. Finally, the DNA fragments were then visualized under UV light. As the result, the DNA fragments for each sample from 2nd well to 13th well as labelled in figure 1 existed as two bands with the same distance in the gel. The sizes of the bands become smaller towards the positive pole of electrical charge. The smaller band would be 752bp whereas the longer band is calculated to be 3609bp. At both end of the sample wells, the 1kb DNA ladder was fully separated into many bands as a standard to identify the approximate size of DNA molecule on gel. …show more content…
Hence, during electrophoresis, the negatively charged DNA fragments would be pulled towards the positive end of machine when the electric current is passed across the agarose gel covered with buffer. Moreover, the agarose gel has three- dimensional mesh structure in its cross linking medium. The DNA molecules would experience the resistance from the agarose mesh during migration. In this way, the DNA molecules with smaller size are able to travel faster through the mesh. Therefore, different sizes of DNA fragments would travel in different speed causing the separation of the bands during electrophoresis (Guruatma,