Restriction enzymes (restriction endonuclease) are enzymes that cut DNA at a specific region of nucleotide sequences, known as the restriction site. To cut the DNA, restriction enzymes make two incisions, one through each strand, or sugar-phosphate backbone. After cutting, another type of enzyme must be able to reattach the DNA back together. This type of enzyme is called the DNA ligase, which facilitates the joining of DNA through catalyzing the formation of phosphodiester bonds.
In molecular cloning, the desired DNA to be cloned is obtained from an organism of interest and treated with enzymes to generate smaller fragments. Then the fragment are joined together with a vector DNA and then introduced into the host organism (i.e. E. coli bacteria) through DNA ligation. Then the host organism will replicate the recombinant DNA along with its own DNA to generate a population of genetically modified organisms. To look at molecular cloning on a molecular level, two different DNA molecules are cut with the same restriction enzyme, results in to compatible sticky ends. The pieces of DNA are isolated, and then re-annealed by DNA ligase. The DNA can then be incorporated into the bacteria and replicated and purified.
This process is applied in this experiment, in which a pi...
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...ern when the lambda DNA is cut by HindIII
The ligation did not go to completion after 1 minute because there are clearly several faint bands below the top band. After 10 minutes, it is safe to say that the fragments have mostly ligated back together. However, the fully ligated DNA looks very different from the uncut DNA. In fact, the intensity of the ligated DNA band is much weaker than that of the uncut DNA. This is most likely be caused by dilution. The uncut DNA sample is much more concentrated than the ligated sample, which would have a more intense peak. Other than peak intensity, they both have the sample size because they are on the same level of the gel.
Overall, the experiment was successful in terms of digesting DNA, ligating DNA, and yielding relatively accurate data. In the future, more times and better quality enzymes can be used to improve the quali
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