Ligation Lab Report

Once the plasmids are cut in a restriction digest reaction, it is then run on an agarose gel. It is important that this is done. The digest gel is used as a confirmation test. It allows one to verify whether or not the restriction enzymes cut the plasmids at a specific sequence; by comparing the base pairs of the product to the 1 Kb ladder (provided one knows the number of base pairs in the expected fragments). One will be able to determine whether the plasmid cut, and if it cut the fragment like it was expected to in day 3 of the experiment (refer to appendix for more information).

Once plasmids are digested and confirmed the next phase of making recombinant DNA is to successfully ligate the fragments. Ligation is the process of sealing sticky ends of plasmids fragments that contain the ampicillin resistant gene or the kanamycin resistant gene. Refer back to Figure 3 for a visual representation of ligation in action. Once ligase is added to the sample, a confirmation test must be done in order to prove ligation successfully occurred. One must remember that ligated plasmids will be enormous. The reasoning being is that, the fragments that were cut by the restriction enzyme where big. Therefore, these

In order to prove that one has successfully combined two plasmid fragments that contained these antibiotic resistant fragments the ligated plasmid must be transformed. Transformation is the uptake of DNA by an organism. As seen in day 6(refer to appendix) of the experiment the organism that incorporated recombinant DNA is E.coli. In order for E.coli to take up Dna it must be competent, meaning that it has the ability to take up DNA. By incubating the plasmids in Cacl2 and heat shocking the cell E.coli became competent. The calcium binds to the negatively charged DNA so that when the cell is heat shocked it makes holes in the plasma membrane that the DNA can slip through. Hence transformation