Solution I is added to cells, followed by solution II and then Solution III. In
ID: 202270 • Letter: S
Question
Solution I is added to cells, followed by solution II and then Solution III. In your own words describe the role of each solution in the protocol. What does it accomplish?
Break open (lyse) the cells containing the DNA of interest. This is done using a solution (cleverly called Solution 1) that contains glucose, Tris, and EDTA. Tris is a buffering agent used to maintain a constant pH (-8.0). EDTA is a divalent ion chelator that helps protect the DNA from degradative enzymes (called DNAases). DNAases require Mg2 for activity, so the EDTA binds the Mg2 ions preventing this cofactor from being available in the solution for the DNAases. Once the cells are resuspended in Solution 1, you then add a second solution called Solution 2 which contains NaOH and SDS (a detergent). Since NaOH is a strong base it causes the solution to become highly alkaline. The alkaline mixture ruptures the cells, and the detergent breaks apart the lipid membrane and solubilizes cellular proteins. NaOH also denatures the DNA into single strands. After step 2 the cells will be lysed Once the cells are lysed, you now need to get rid of the lipids and proteins present in the sample Precipitation of the protein is aided by the addition of a salt such as ammonium or sodium acetate. At this step you will add a solution called guess what Solution 3. Solution 3 contains a mixture of acetic acid and potassium acetate. The acetic acid neutralizes the pH, allowing the DNA strands to renature The potassium acetate salt precipitates the SDS from solution, along with the cellular debris (e.g proteins). The E. coli chromosomal DNA, a partially renatured tangle at this stage will get trapped in the precipitate. Only the plasmid DNA will remain in solution The next step is to separate the plasmid DNA from the cellular debris and E. coli chromosomal DNA. This is accomplished by placing the tubes containing your sample into a microcentrifuge and centrifuging the tubes for 10 minutes. The insoluble cell debris will pellet to the bottom of the tube, and the plasmid DNA will remain in solution The next step is to carefully transfer the supernatant to a fresh microcentrifuge tube using a pipetman with a clean tip. Be very careful to avoid taking any of the white precipitate during the transfer. It is better to leave a little supernatant behind to avoid taking the precipitate The next step is to concentrate the DNA into a smaller volume. This is accomplished by precipitation of the DNA with Isopropanol. Isopropanol effectively precipitates nucleic acids, but is much less effective with protein precipitation. Aquick precipitation can not only concentrate the DNA, but also provide another level of purification from protein contaminants. If allowed to site for longer time, proteins will also precipitate. Thus, this step is relatively quick to prevent protein precipitation After incubation of the DNA in isopropanol for 10 minutes at room temperature, the tubes containing the DNA are again placed in the microcentrifuge and centrifuged for 10min. After centrifugation, a milky white pellet should be at the bottom of the tube. Carefully pour off the supernatant without dumping out the pellet. Drain the tube on a paper towel and allow the tube to dry for 15 minutes Your TA will tell you when it is sufficiently dry When the dry pellet is ready, add 20 microliters of a solution called TE to the tube. TE stands for Tris EDTA. Again the Tris is the buffer and the EDTA is to protect the DNA from degradation 1. 2. 3. 4. 5. 6. 7. 8.Explanation / Answer
Solution 1-3 are employed to isolate plasmid DNA from E.coli.
Solution 1 contains glucose, Tris and EDTA. This solution is used during plasmid DNA extraction to help lyse the cell and nuclear membranes in order to release the DNA. DNA is not accessible as cells are covered by the cell membrane and a nuclear envelope. DNA is present in a complexed form with histones as chromosomes in a cell. Hence, the plasma membrane and nuclear envelope should be removed so that DNA can come into the solution. Tris maintains the pH of the solution to 8. DNA is sensitive to pH changes. At pH 8, the DNA will be in a polar aqueous state as the phosphodiester bonds are negatively charged. EDTA is a chelator of Magnesium ions. Magnesium ions are required for the activity of DNases present in the cell. DNases break down DNA into constituent nucleotides. When cells undergo lysis, the DNAses present in cytoplasm are released. Hence, DNA has to be protected from them, otherwise the DNA will degrade. Glucose is added to create an osmotic gradient in the cell. There is high concentration of glucose outside the cell and low concentration inside the cell. This difference in concentration will create an osmotic pressure on cell membrane. The cells will now be in a flabby state and can be lysed by solution 2.
Solution 2 contains sodium hydroxide (NaOH) and detergent sodium dodecyl sulphate (SDS). Sodium hydroxide raises the pH to 8-12 (alkaline). As a result, the proteins are denatured due to high pH. DNA will separated as it is denatured. The SDS added will solubilize proteins as it can bind to them and give them a negative charge. SDS will also solubilize the phospholipids present in the cell membrane, thereby causing lysis. After addition of solution 2, the cells will lyse releasing the DNA in solution.
Solution 2 had raised to pH to alkaline range. In order to neutralize the pH, solution 3 is added that contains acetic acid and potassium acetate. Acetic acid will reduce the pH to a neutral range. This allows DNA present as single strands to anneal and form double stranded DNA. Potassium acetate will precipitate the proteins and lipids present in solution. SDS along with potassium will form an insoluble precipitate. Chromosomal DNA from E coli is large and partly annealed at this stage. Hence, chromosomal DNA will be trapped in the precipitate. The circular plasmid DNA due to its smaller size will remain in solution. The precipitate is removed from solution in next step, to purify the plasmid DNA.
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