Question

In: Biology

Using the alkaline lysis protocol, explain how the following scenarios would affect the experimental outcome: a....

Using the alkaline lysis protocol, explain how the following scenarios would affect the experimental outcome:

a. 100uL of solution 1 was added to a cell pellet, but the pellet was not re-suspended

b. Solution 3 was made without potassium/sodium acetate

Solutions

Expert Solution

Answer: a. If the pellet is not resuspended in the solution 1 when plasmid isolation is performed using an alkaline lysis method there are chances that you may see low quality plasmid, RNA contamination and low yield of plasmid. Now, this can happen because cells are not exposed to the active content of solution I i.e. EDTA and RNAse. EDTA chelates the divalent ions which are required for the activity of DNAse which degrades the plasmid and also for the integrity of the cell membrane. EDTA inactivates DNAse and destabilizes the cell membrane. Failure to suspend pelleted cells in the solution I may affect the overall quality and quantity of isolated plasmid and it can also affect the efficacy of other two steps in plasmid isolation.
b. Potassium acetate causes protein and the SDS complex to settle down in the mixture so the resulting solution appears somewhat transparent after addition of solution 3. The most important function of potassium acetate is that it renatures the plasmid but the genomic DNA is not able of re-nature and hence it settles down along with protein precipitate so in the supernatant only plasmid is left. If solution three does not contain potassium acetate we won't be able to precipitate protein and genomic DNA and plasmid will not renature. The resulting solution will be very turbid and white in color and when you run that into agarose get you will hardly see any bands of the plasmid.
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