The goal is to import a vector into the genome of the host cells. The vector in this case coded for ampicillin resistance, GFP and the LAC operon. The operator we are interested in is LAC, the ampicillin resistance is used to kill off any cells which did not uptake the vector and expression of GFP allows visual observation of successful transformation. The plasmid was introduced by heatshock.

IPTG activates the LAC operon, so all plates contained IPTG. The schematic below shows the procedure of checking for succesful transformation, and the controls.

We do not expect to observe any fluorescent cells in the plates one and two because, there is no plasmid DNA for the cells to uptake. These are the two controls to check that the host cells, do not fluoresce by themselves. There should be fluorescent cells in the plate 3, because there is plasmid DNA present. However, since we are not selecting for the transformed cells, the wild type (non-fluorescent) will be dominant and probably drown out any mutants. Plate 4 should contain only fluorescent cells, because all wild type cells will be killed off by the ampicillin. (Note: there might be spotaneous or random mutations in the host cells that gives rise to ampicillin resistance but this has a very low probability)

The results are as we expected, the only bad point is that the number of cells expressing the vector is very low!

The aim of this experiment was to perform an enzymatic digest on the Lambda phage genome. The results will be visualised using gel electrophoresis (agarose gel). From the New England Biolabs website (http://www.neb.com) it is possible estimate the number of cleavage sites for the respective enzymes and predict how many bands are expected on the gel. In face, since the Lambda phage genome has been studied thoroughly, it will be possible to estimate how large these fragments should be and hence their position on the gel.

This image shows where in the Lambda Genome hindIII and ecoRI cut.

This is the original, unprocessed gel.

Notes:
The image was cropped, rotated, converted to gray scale and then the brightness and contrast were altered.

The gel seems to have worked to some degree: the only major problem being the ladder not giving clear bands. The EcoRI digest appears to have worked well, almost four out of the five expected bands are visible. Two of the fragments are of very similar size so a double band is expected - this is the third band down on the gel, hence the 'almost'. Only the fifth and heaviest band does not appear as expected. Though there does appear to be a much larger fragment roughly halfway down the gel, which cannot be explained. Viusualisation of the HindIII digest was not quite so successful: only three out of the six expected bands are clear. The three lightest fragments show up, the three heavier ones are not visible and once again there is a rogue band half way down the gel. From the streaking patterns the lane appears to have been punctured or loaded with too much DNA.

All predicted gels were sourced from the New England Biolabs website (http://www.neb.com) The predicted gels have been altered because they did not take into account the circular nature of Lambda phage DNA.