Nucleic Acid Extraction
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microbial, animal, plant
Types of samples fit for extraction
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Microbial
Whole culture, single
colony, soil, water
colony, soil, water
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Animal
Blood, fresh or preserved
tissue, bodily fluids, feces
tissue, bodily fluids, feces
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Plant
Leaves, roots, bark, fruit,
seed
seed
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Genomic DNA, Extrachromosomal DNA (pDNA), Mitochondrial DNA, Nuclear RNA, Cytoplasmic RNA
Where can we find DNAandRNA?
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Lysis, Precipitation, Purification, Elution, Quantitation, Storage
Nucleic acid extraction steps
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Lysis
To free the NA from the cell,
may involve enzymes
may involve enzymes
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Precipitation
Separate NA from cell
debris
debris
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Purification
Enzymatic treatment to
remove DNA or RNA
remove DNA or RNA
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Elution
Retrieval of NA in buffer
or NFW
or NFW
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QuantitationExtraction
methods
methods
UV-Vis
Spectrophotometry
Spectrophotometry
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Storage
RNA in -80ºC
DNA in -20ºC
DNA in -20ºC
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Phenol-chloroform extraction, Spin column-based extraction, Magnetic bead extraction, Alkaline lysis (for pDNA)
Extraction
methods
methods
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Phenol-chloroform extraction
is a technique used in molecular biology to purify nucleic acids, primarily DNA and RNA, from cellular components.
It involves mixing a sample with a mixture of phenol, chloroform, and sometimes isoamyl alcohol, which creates two distinct phases (aqueous and organic) after centrifugation.
Proteins and other cellular debris are partitioned into the organic phase, while nucleic acids remain in the aqueous phase, allowing for their separation and purification
It involves mixing a sample with a mixture of phenol, chloroform, and sometimes isoamyl alcohol, which creates two distinct phases (aqueous and organic) after centrifugation.
Proteins and other cellular debris are partitioned into the organic phase, while nucleic acids remain in the aqueous phase, allowing for their separation and purification
15
Spin column-based extraction
a common molecular biology technique used for purifying nucleic acids (DNA or RNA) and proteins. It involves using a solid matrix, usually silica, within a column to selectively bind the target molecule from a sample.
The sample is then passed through the column, allowing other components to flow through while the target molecule remains bound.
Finally, the target molecule is eluted from the column with a specific buffer.
The sample is then passed through the column, allowing other components to flow through while the target molecule remains bound.
Finally, the target molecule is eluted from the column with a specific buffer.
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Magnetic bead extraction
a method used to isolate nucleic acids (DNA or RNA) by utilizing magnetic beads that bind to the target molecules.
These beads, with a paramagnetic core, are added to a sample, and a magnetic field attracts them, allowing for separation of the beads (and bound nucleic acids) from other cellular components.
These beads, with a paramagnetic core, are added to a sample, and a magnetic field attracts them, allowing for separation of the beads (and bound nucleic acids) from other cellular components.
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Alkaline lysis (for pDNA)
a method used in molecular biology to isolate plasmid DNA from bacterial cells.
It involves lysing bacterial cells and denaturing the DNA, selectively separating plasmid DNA from chromosomal DNA due to the differential stability of the two under alkaline conditions.
It involves lysing bacterial cells and denaturing the DNA, selectively separating plasmid DNA from chromosomal DNA due to the differential stability of the two under alkaline conditions.
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Tris-NaCl-EDTA buffer, Phenol:chloroform:isoamyl alcohol, Guanidium thiocyanate, CTAB (Cetyltrimethyl ammonium bromide), CaCl2, Proteinase K, DNase I
Commonextraction reagents
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1. Collect approx. 3 mL of saliva from a volunteer. Stimulate salivary glands by
massagin them for 1 to 2 minutes. Remember to rub your tongue around your
mouth as you do this in order to agitate and loosen cells from your mouth.
2. Add 3 mL of 10% dishwashing liquid to the saliva and gently swirl or invert 5
times. Let stand for 5 minutes.
3. Add 3 mL of 10% salt to the solution. Gently swirl or invert the tubes for ~ 30
seconds.
4. Add 4 mL of isopropyl alcohol by slowly and gently pouring it down the side of
the tube. Let it stand for 5 minutes. You should be able to see strands of your
DNA forming.
massagin them for 1 to 2 minutes. Remember to rub your tongue around your
mouth as you do this in order to agitate and loosen cells from your mouth.
2. Add 3 mL of 10% dishwashing liquid to the saliva and gently swirl or invert 5
times. Let stand for 5 minutes.
3. Add 3 mL of 10% salt to the solution. Gently swirl or invert the tubes for ~ 30
seconds.
4. Add 4 mL of isopropyl alcohol by slowly and gently pouring it down the side of
the tube. Let it stand for 5 minutes. You should be able to see strands of your
DNA forming.
Extract Nucleic Acids at Home
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