Dna Gel Electrophoresis Lab

Introduction:

Gel electrophoresis is a method used to separate DNA fragments based on size and charge. This technique uses a gel that acts like a filter with pores in which molecules travel through. There are two types of gel which can be used depending on the molecules being separated, polyacrylamide gel or agarose gel (Karcher, 1993). In this gel electrophoresis, 0.8% agarose gel is used (Rose, 2010). The gel is subjected to an electrical current that forces the DNA with a negative charge to move through the gel to the positive end. The smaller fragments travel faster than larger fragments. So the sizes of the fragments can be distinguished through the mobility rate. The bands of the fragments are seen by the ethidium bromide (EtBr) which is fluorescent dye (Rose, 2010). Ethidium bromide reacts and intercalates between the nucleotide bases of the DNA molecules. The ethidium bromide fluoresces more in UV light. At the end a gel picture is taken which is used as the restriction map.

In this experiment, the linear double stranded DNA is obtained from a bacterial virus, bacteriophage λ (phage λ), which has a genome of 50 000 base pair in size (Rose, 2010). The shorter fragments of the DNA are produced by the restriction enzyme that cuts at recognition sites in the DNA. Hind III was used as a marker to determine the sizes of the fragments cut by restriction enzymes BamH1 and EcoR1. The observed fragments sizes and the distance travelled are used to plot a standard graph. The standard graph of the Hind III determines the sizes of the DNA bands produced By EcoR1 and BamH1 (Rose, 2010). So the purpose of the lab is to determine the size of the fragment by using the distance travelled.

Discussion:

In this experiment restriction enzymes Hind III, EcoR1, and BamH1 were used to cleave the DNA at the restriction sites to produce smaller fragments. These fragments were run through the gel electrophoresis and which produced bands that were visible on the gel. These bands were measured from the origin of migration to the leading edge. The DNA digested with Hind III, which is lane 1 of all the 4 gels that serves as the standard curve to compare the other DNA bands. In all the 4 gels which ever lane 1 produced the most visible bands were used as to measure the distances, in this case gel 1 was used (Table 1). These measurements were used to make the standard curves (Figures 1 & 2). Then lane 2 of all the 4 gel is the uncut λ DNA. The other 6 lanes of gel 1 and 2 were cut by EcoR1, and which ever lane produced the best bands were used to measure distance. The lane producing the most visible bands is from lane 5 in gel 2 (Table 2). In gel 3 and 4, the other 5 lanes were cut by BamH1. For BamH1, the most visible bands were in lane 5 of gel 4. They fragment sizes of BamH1 and EcoR1 were extrapolated from the standard curve (Figure 1). Eco R1 was