Restriction Enzyme Digestion

The aim of this experiment was to investigate the effects of methylation on digestion of genomic DNA with different restriction enzyme concentrations.

We performed a genome prep assay on 2 different strains of E.Coli - a Dam positive strain (TOP10) and a Dam negative strain (GM2163). The DNA of the Dam positive strain is naturally methylated, whilst the DNA of the Dam negative strain was unmethylated. This allowed comparisons to be drawn between the amount of cleavage by restriction enzymes.

A restriction digest was performed using our 2 restriction enzymes (DpnII and TaqI) each at varying concentrations. The results can be seen below:

DpnII Restriction Digest

Here we can see clearly the differences between the Dam positive and negative strains.
The Dam-ve strains are fully cleaved at all different concentrations of restriction enzyme. This produces the 'smear' that can be seen, as the DNA fragments are all of varying, but short lengths. This smear is not present in the control without the restriction enzymes. Good contrast can be seen against the Dam+ve genome, of which increasing amounts of cleavage can be seen with increasing restriction enzyme concentration. At 0.2ul restriction enzyme, the cleavage is minimal, and the gel profile is similar to the negative control. At 1.0ul restriction enzyme, there is significantly more cleavage, and the fragments are beginning to form the 'smear' effect seen in the Dam -ve strain.

DpnII Restriction Digest

Again, there are clear differences between the Dam positive and negative strains.