MCAT Biology : Compound Purification
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Example Questions
Example Question #1 : Compound Purification
To isolate DNA from a cell pellet, a student is performing an extraction using phenol and chloroform. The cells were lysed with 0.5mL of lysis buffer. 0.5mL extraction solution, consisting of phenol:chloroform:isoamyl alcohol (25:24:1), was added to the lysate. The sample was centrifuged at high speed for five minutes. The liquid in the tube was separated into three phases after centrifugation (see diagram).
In which layer will nucleic acids (RNA and DNA) be found?
1
3
Cannot be determined
2
1
RNA and DNA are hydrophilic substances, and will be found in the aqueous layer after the cells are lysed. Based on molecular weights of water (18), phenol (94), and chloroform (119), it is evident that the aqueous layer will be on the top, denoted as layer 1.
Example Question #2 : Compound Purification
To isolate DNA from a cell pellet, a student is performing an extraction using phenol and chloroform. The cells were lysed with 0.5mL of lysis buffer. 0.5mL extraction solution, consisting of phenol:chloroform:isoamyl alcohol (25:24:1), was added to the lysate. The sample was centrifuged at high speed for five minutes. The liquid in the tube was separated into three phases after centrifugation (see diagram).
In which layer will lipids and other cellular debris be found?
1
2
3
Cannot be determined
3
Lipids and other cellular debris are hydrophobic, and can be dissolved in the organic (bottom) layer. We know that the organic solvents will settle in layer 3 due to their relative densities compared to water. Phenol and chloroform are heavier molecules than water, with molecular weights of 94 and 119 respectively, and will thus be found in the bottom layer.
Example Question #44 : Organic Analyses And Lab Techniques
To isolate DNA from a cell pellet, a student is performing an extraction using phenol and chloroform. The cells were lysed with 0.5mL of lysis buffer. 0.5mL extraction solution, consisting of phenol:chloroform:isoamyl alcohol (25:24:1), was added to the lysate. The sample was centrifuged at high speed for five minutes. The liquid in the tube was separated into three phases after centrifugation (see diagram).
What is the nature of each phase?
1: Aqueous
2: Interphase
3: Organic
1: Phenol
2: Chloroform
3: Isoamyl alcohol
1: Phenol
2: Isoamyl alcohol
3: Chloroform
1: Organic
2: Interphase
3: Aqueous
1: Chloroform
2: Isoamyl alcohol
3: Phenol
1: Aqueous
2: Interphase
3: Organic
The purpose of a phenol:chloroform extraction is to separate organic and aqueous cellular components. The lysis buffer is an aqueous solution, while the phenol:chloroform extraction solution is organic. Generally, organic solutions are more dense that aqueous solutions, resulting in the aqueous solution being on the top and organic being on the bottom. The interphase consists of proteins containing both hydrophilic and hydrophobic domains.
You can also determine relative density by calculating the molecular weights of the solvents. Doing so will show that water (MW = 18) is much lighter than phenol (MW = 94) or chloroform (MW = 119).
Example Question #45 : Organic Analyses And Lab Techniques
To isolate DNA from a cell pellet, a student is performing an extraction using phenol and chloroform. The cells were lysed with 0.5mL of lysis buffer. 0.5mL extraction solution, consisting of phenol:chloroform:isoamyl alcohol (25:24:1), was added to the lysate. The sample was centrifuged at high speed for five minutes. The liquid in the tube was separated into three phases after centrifugation (see diagram).
In which layer will proteins be found?
Cannot be determined
2
Both 1 and 3
1
3
2
Proteins generally contain both hydrophobic and hydrophilic domains, and therefore, do not completely associate with either the organic or aqueous layers. They can be found in the interphase layer, denoted as layer 2. This allows different domains of the protein to interact with each phase. Hydrophilic regions will interact with the aqueous phase (layer 1), while hydrophobic regions will orient toward the organic phase (layer 3).
Example Question #46 : Organic Analyses And Lab Techniques
To isolate DNA from a cell pellet, a student is performing an extraction using phenol and chloroform. The cells were lysed with 0.5mL of lysis buffer. 0.5mL extraction solution, consisting of phenol:chloroform:isoamyl alcohol (25:24:1), was added to the lysate. The sample was centrifuged at high speed for five minutes. The liquid in the tube was separated into three phases after centrifugation (see diagram).
The student saves the layer containing nucleic acids and transfers it to a new tube. The student then adds ethanol and salt, and centrifuges the solution at high speed. Where is the DNA in this new solution after centrifugation?
Pellet
Organic layer
Supernatant
DNA cannot be isolated by adding ethanol
Interphase
Pellet
After the first extraction, the DNA will be found in the aqueous phase due to its negative charge and hydrophilic nature. Transferring the aqueous phase (with DNA) and adding ethanol and salt will not result in an extraction; there will only be one phase after centrifugation: the aqueous phase. Following centrifugation, the DNA and salt precipitate from the aqueous ethanol solution and form a DNA pellet.
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