The objective of this experiment was to reassert that mutation of PUC19 really does occur. The PUC19 sample was obtained from an E. coli bacteria whose LacZ gene had been mutated and hence it was non functional. Production of β-galactosidase had, therefore, stopped. The main idea of this experiment was to reverse this mutation so that the gene can regain its ability to produce β-galactosidase. The production of β galactosidase is indicated by presence of blue colonies on the plate which results from the cleavage of X-galactosidase which is colorless into galactosidase which is blue in color.
The Puc19M was heated to denature the double stranded DNA and placed on a tube containing ice. An annealing buffer, together with a mutagenesis primer was added during which, the annealing buffer helped in bringing the primer and the single strand DNA into consistency. The mutagenesis primer was added to perform reverse functions to the mutated gene in order to make it functional. A selection primer was also added to alter the restriction site of the plasmid thus allowing the synthesis of a new circular DNA strand.
A synthesizing buffer was added to provide the suitable environment required for the synthesis of the new DNA strand. The addition of T4 DNA polymerase was to facilitate the hybridization of the old and the new DNA strands. T4 DNA ligase was as well used to join the two ends of the new DNA strand. The DNA obtained was introduced into E.coli mutS since they lack the gene responsible for repair of mismatched sequences. Hence, the new DNA strand could not be repaired.
After electroporating the mixture overnight, a pellet was formed onto which, a cell suspension was added to prevent enzymatic reactions. The cell was lysed to obtain the plasmid DNA. Addition of an alkaline solution was to get rid of all the membrane proteins that could have been present. The solution was then neutralized.
The column that had been prepared during centrifugation was inserted into a tube as the supernatant was discarded so that the DNA could be left stuck in the resin. Ethanol was used to dissolve away any impurities but after the solution had been centrifuged two more times. Centrifugation was performed one more time to further remove any unwanted materials. This was followed by the transfer of the spin into a smaller centrifuge tube where water that had been freed of nuclease, was added to wash away resin from the DNA. This process was carried out in the microcentrifuge tube for one minute.
A NdeI digest procedure, using the newly formed DNA, was then carried out. The new DNA was used because, unlike the old DNA strand, it does not have restriction sites for NdeI. The procedure involved addition of the plasmid DNA sample into a buffer containing sterilized water and incubating the solution for 2 hours.
Due to the lack of restriction site for NdeI in the new DNA strand, the new Puc19 will remain in its circular form and as a result, the LacZ genes present in the Puc19 will still be active due to the lack of NdeI restriction site which would have, otherwise, hindered the functioning of the LacZ genes. As a result, the ability of the genes to produce β galactose would be restored which, as discussed earlier, was evidenced by the appearance of blue colonies on the plate.