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[full-credit student answers, with comments]
1. List the two steps of the "midiprep" procedure that serve to eliminate cellular proteins. For each step, provide a brief explanation of how proteins would be separated from plasmid DNA.
Steps 4-5 and 10 serve to separate the cellular proteins from our plasmid DNA. Steps 4-5 precipitate and pellet out the cellular proteins while the plasmid DNA remains in the supe. Step 10 utilizes the properties of DNA and the anion exchange resin to further isolate plasmid DNA from protein. Buffer QC contains 1.0 M NaCl and a pH of 7.0. Both these factors aid in the elution of protein while the plasmid DNA remains adhered to the QIAGEN tip.
2. List the two steps of the "midiprep" procedure that serve to eliminate RNA. For each step, provide a brief explanation.
Steps 2-3 and 10 isolate the DNA from RNA. In step 2 RNaseA is added. However, it has no immediate effect because it is only on the outside of the cells. When the 1% SDS detergent is (in step 3) added it lyses the cells and exposes the cellular RNA to the RNaseA. In step 10 the buffer QC performs the same function on RNA as it does for protein: the RNA is eluted through the column under the same conditions.
[RNA would not be expected to precipitate out of solution in step 4, with the genomic DNA and denatured protein. The RNase digestion does serve to fragment the RNA into tiny pieces, though, which do not bind with high affinity (or at all) to the QIAGEN tip. Those fragments that do bind are washed off in buffer QC in step 10.]
3. How is genomic DNA separated from plasmid DNA in this procedure? (You may wish to refer back to previous labs to answer this question). In order for this separation to work, what must the experimenter be careful to avoid?
Plasmid DNA is separated from the genomic DNA due to the nature of its structure. When the pH of the solution is reduced in the alkaline lysis step (step 3), the H-bonds of both plasmid and genomic DNA are melted. However, since the plasmids are covalently-closed supercoils, the 2 strands in the plasmid remain linked. Upon restoring neutral pH the plasmid DNA sponteneously reanneals. The genomic DNA, however, is permanently denatured and precipitates out with the cell lysate. The experimenter must carefully avoid shearing of the DNA, usually by mixing too hard; furthermore, it is essential that the midiprep is not left in buffer P2 (SDS/NaOH) too long, otherwise permanently denaturing the plasmid DNA.
[Genomic DNA is also generally associated with the plasma membrane/cell wall components. This association facilitates its precipitation.]
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Created by:
bkbaxter@lclark.edu
Updated: 21 November 00