Lab 6: Isolation of Chromosomal DNA
Mic 428L/ Section 001
In biological research to address and eventually answer a multitude of questions, usually involves isolating chromosomal DNA. The purpose in this particular lab was to isolate chromosomal DNA from mutants grown and observed in lab 5 and then digest the DNA using a restriction enzyme. The fragments left from digestion will be ligated and then transformed into a strain of E. Coli DH5αλpir containing the pir gene pi product for replication. This gene will allow only the plasmid containing the Tn to replicate. Afterward, the plasmid was isolated and sequenced into adjacent chromosomal DNA to determine which gene was interrupted by the Tn. The isolation of genomic DNA is a prerequisite for many types of experiments including DNA sequencing and cloning. One common goal of the isolation of the chromosomal DNA is to avoid shearing. The large chromosome is fragile and can be sheared very easily. Although there will always be some chromosomal DNA that cannot avoid shearing, most methods use a gentle lysis step using a detergent, SDS, and a proteinase enzyme to degrade proteins. Chloroform will also be used in the isolation procedure to remove any additional proteins bound to the DNA that we do not want. Additionally, the chromosomal DNA will precipitate out using a non polar solvent, isopropanol or ethanol. DNA is polar because of the negative charge on its phosphate backbone. Therefore, when it is exposed to a less polar solvent, such as ethanol or isopropanol, it will reduce the amount of surface area that is exposed to that solvent therefore it will form a ball and precipitate out. The chromosomal DNA will be digested with a restriction enzyme once it has been isolated. We will use the restriction enzyme BamHI which cuts in fragments of around 5,000 base pairs. This means that BamHI will digest the chromosome once every 5,000 base pairs. For a genome of 6 Mb, BamHI will digest it into...
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