Question

You decide you need more of the DNA to work with than what you were given, but do not want to ask your colleague for more. You therefore ligate the cDNA into a high copy number plasmid and isolate the recombinant DNA molecule. For your extraction, you use the QIA prep miniprep procedure, which is based on alkaline lysis of bacterial cells followed by adsorption of DNA onto silica in the presence of high salt. You will have your technicians carry out the prep and thus ask them to review the protocol and report to you on the following questions.

A. Why is there RNaseA in Buffer P1 (resuspension buffer) and when does it actually carry out its intended role?
B. What is the purpose of having SDS and NaOH in Buffer P2? In other words, what does each do?
C. What happens to chromosomal DNA and other cellular debris when Buffer N3 is added?

Answer 1

RNase A in Buffer P1 degrades RNA during plasmid DNA purification. SDS and NaOH in Buffer P2 lyse bacterial cells and denature DNA. Upon adding Buffer N3, it precipitates cellular debris, genomic DNA, and proteins, which can be removed by centrifugation.

Step-by-step explanation:

Purification of Plasmid DNA from Bacterial Cells

In the process of plasmid purification, there are specific reasons for each of the components used in the buffers. RNase A is included in Buffer P1, known as the resuspension buffer, to degrade any RNA that may be present, ensuring that the final product is devoid of RNA contamination. This enzymatic reaction takes place during the resuspension of the bacterial pellet, when RNase A effectively degrades RNA.

Buffer P2 serves the purpose of lysing the bacterial cells to release plasmid DNA. It contains SDS, a detergent that disrupts the cell membrane, and NaOH, a strong base that denatures the chromosomal and plasmid DNA by causing their strands to separate.

Lastly, when Buffer N3 is added, it neutralizes the pH and assists in the formation of complexes with SDS, genomic DNA, and proteins. These complexes, along with other cellular debris, are precipitated out, allowing for their subsequent removal by centrifugation. The clear supernatant containing the plasmid DNA can then be transferred to a clean tube for further purification steps.

A. There is RNaseA in Buffer P1 (resuspension buffer) in order to degrade any RNA molecules present in the sample. RNaseA is an enzyme that specifically breaks down RNA. It carries out its intended role when Buffer P1 is added to the sample, effectively degrading any RNA and leaving only the DNA behind.

B. The purpose of having SDS and NaOH in Buffer P2 is to break down the bacterial cell membrane. SDS (sodium dodecyl sulfate) is a detergent that disrupts the integrity of the cell membrane, while NaOH (sodium hydroxide) helps to denature proteins and DNA. Together, they facilitate the release of the DNA from the cells.

C. When Buffer N3 is added, it causes the precipitation of chromosomal DNA and other cellular debris. This precipitate can be easily separated from the desired plasmid DNA by centrifugation or filtration, resulting in the purification of the plasmid DNA.