Minipreps have become quite commonplace in laboratories, and kits have become av
ID: 51231 • Letter: M
Question
Minipreps have become quite commonplace in laboratories, and kits have become available containing the appropriate buffers needed to perform the plasmid extraction/purification. The kit will often give a shorthand name (e.g. P1) to these buffers and provide a step-by-step protocol. However, it is important to know what is inside each of these buffers and what the purpose of each solution is. For each of the following buffers, provide a more descriptive name, and describe the purpose of each of the main ingredients listed. Use your own words and cite sources if necessary. An example has been done for you. (20 points)
Buffer EB: Elution Buffer
Tric-Cl, pH 8.5: The purpose of the Tris is to buffer the pH to keep it within the
optimal range for maximal DNA elution off of the silica column, which is between
pH 7-8.5. Note that also water can be used in lieu of the buffer ‘EB’ because a
low-salt solution such as water will also elute the DNA off of the silica efficiently.
Buffer P1:
EDTA:
RNAse:
Tris-Cl:
Buffer P2:
NaOH:
SDS:
Buffer N3:
Sodium or Potassium acetate, pH5.5:
Guanidine hydrochloride:
Buffer PE:
Ethanol:
Explanation / Answer
Buffer P1 is the resuspension buffer, which contains 50mM Tris-Cl, pH 8.0, 10 mM EDTA, and 100 µg/ml RNase A. This buffer has to be stored at -40C after adding RNase A. It is used during purification of plasmid DNA.
EDTA: It chelates the divalent cations in the solution and prevents the damage of DNA by DNases.
RNase A: It separates the RNA from the cell extract by breaking the RNA.
Tris-Cl: It acts as a buffering agent and maintains the pH of resuspension buffer at 8.0.
Buffer P2 is the lysis buffer, which contains 200 mM NaOH, and 1 % SDS. This buffer can be stored at room temperature.
NaOH: It denatures the DNA, which involves conversion of double stranded DNA (dsDNA) into single stranded DNA (ssDNA). It also breaks down the cell wall.
SDS: It destroys and solubilizes the cell membrane. It also denatures the proteins in the cell. Thus, the proteins are removed as precipitate.
Buffer N3 is the neutralization buffer. This buffer can be stored at room temperature.
Sodium or Potassium acetate, pH5.5: It renatures the plasmid DNA (ssDNA – dsDNA).
Guanidine hydrochloride: It precipitates the cell debris and chromosomal DNA.
Buffer PE is the wash buffer. This buffer can be stored at room temperature.
Ethanol: The addition of ethanol inhibits the action of enzymes and other chemicals that were added earlier like RNase, EDTA, and some salts.