Embed Size (px)
Citation preview
DNA extraction
DNA is building block of the life for all living creatures
Every thing from bacteria to human has DNA in their cellular stucture
Every thing living contain DNA DNA extraction is a routine procedure to
collect DNA for subsequent molecular or forensic analysis
Structure of the cell
Physical Characteristics of DNA DNA absorbs UV light at 260 nm Allows
quantitation DNA is water soluble DNA precipitates in alcohols DNA carries a net negative charge DNA has characteristic melting amp annealing
temperatures
Why extract DNAThe isolation of the DNA from biological sample is an essential step in the DNA technology (PCR - RFLP- cloning - hyberdization all this approaches require DNA as template
bulldisease diagnosis bullDNA sequencing
bullgenetically modified organisms (GMO) - agriculture
pharmaceutical
Nucleic Acid PreparationSample Source
Whole blood Buffy coat Serum or plasma Bone material Buccal cells Cultured cells
Amniocytes or amniotic fluid
Dried blood spots Fresh or frozen tissue
(biopsy material) Sputum urine CSF or
other body fluids Fixed or paraffin-
embedded tissue
How to isolate DNAThey are many types of methods to isolate DNA depending
on the type of the sample and the purpose from extraction (organic method (phenol ndash chloroform )-chlex ndashmethanol - commercial kit (FTA)
In genral all type of methods are following three coral steps
1-lyses cell wall by lyseis buffer
2-precipitation of proteins
3 -precipitation of DNA
1-Breaking the cells open commonly referred to as cell disruption to expose the DNA within
2-Removing membrane lipids by adding a detergent
Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents
Lysis bufferLysis buffer bull 50 mM Tris-HCI pH 80 to maintain the pH
of the solution at a level where DNA is stable 1 SDS to break open the cell and nuclear membranes allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins making them more susceptible to protease cleavage)
Precipitating the DNA with an alcohol mdash usually ethanol or isopropanol Since DNA is insoluble in these alcohols it will aggregate together giving a pellet upon centrifugation
DNA extraction ndash the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
bullSDS bullCTAB
bullProteinase K
bullFreezingbullGrinding
Chemical
Enzymatic
Mechanic
Alcohol bullEthanolbullIso-propanol
bullPhenolbullchloroform
bullSodium chloridebullSodium acetate
bullMembranebullBeads
Organic solvents
Salt
DNA binding
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
DNA is building block of the life for all living creatures
Every thing from bacteria to human has DNA in their cellular stucture
Every thing living contain DNA DNA extraction is a routine procedure to
collect DNA for subsequent molecular or forensic analysis
Structure of the cell
Physical Characteristics of DNA DNA absorbs UV light at 260 nm Allows
quantitation DNA is water soluble DNA precipitates in alcohols DNA carries a net negative charge DNA has characteristic melting amp annealing
temperatures
Why extract DNAThe isolation of the DNA from biological sample is an essential step in the DNA technology (PCR - RFLP- cloning - hyberdization all this approaches require DNA as template
bulldisease diagnosis bullDNA sequencing
bullgenetically modified organisms (GMO) - agriculture
pharmaceutical
Nucleic Acid PreparationSample Source
Whole blood Buffy coat Serum or plasma Bone material Buccal cells Cultured cells
Amniocytes or amniotic fluid
Dried blood spots Fresh or frozen tissue
(biopsy material) Sputum urine CSF or
other body fluids Fixed or paraffin-
embedded tissue
How to isolate DNAThey are many types of methods to isolate DNA depending
on the type of the sample and the purpose from extraction (organic method (phenol ndash chloroform )-chlex ndashmethanol - commercial kit (FTA)
In genral all type of methods are following three coral steps
1-lyses cell wall by lyseis buffer
2-precipitation of proteins
3 -precipitation of DNA
1-Breaking the cells open commonly referred to as cell disruption to expose the DNA within
2-Removing membrane lipids by adding a detergent
Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents
Lysis bufferLysis buffer bull 50 mM Tris-HCI pH 80 to maintain the pH
of the solution at a level where DNA is stable 1 SDS to break open the cell and nuclear membranes allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins making them more susceptible to protease cleavage)
Precipitating the DNA with an alcohol mdash usually ethanol or isopropanol Since DNA is insoluble in these alcohols it will aggregate together giving a pellet upon centrifugation
DNA extraction ndash the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
bullSDS bullCTAB
bullProteinase K
bullFreezingbullGrinding
Chemical
Enzymatic
Mechanic
Alcohol bullEthanolbullIso-propanol
bullPhenolbullchloroform
bullSodium chloridebullSodium acetate
bullMembranebullBeads
Organic solvents
Salt
DNA binding
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Structure of the cell
Physical Characteristics of DNA DNA absorbs UV light at 260 nm Allows
quantitation DNA is water soluble DNA precipitates in alcohols DNA carries a net negative charge DNA has characteristic melting amp annealing
temperatures
Why extract DNAThe isolation of the DNA from biological sample is an essential step in the DNA technology (PCR - RFLP- cloning - hyberdization all this approaches require DNA as template
bulldisease diagnosis bullDNA sequencing
bullgenetically modified organisms (GMO) - agriculture
pharmaceutical
Nucleic Acid PreparationSample Source
Whole blood Buffy coat Serum or plasma Bone material Buccal cells Cultured cells
Amniocytes or amniotic fluid
Dried blood spots Fresh or frozen tissue
(biopsy material) Sputum urine CSF or
other body fluids Fixed or paraffin-
embedded tissue
How to isolate DNAThey are many types of methods to isolate DNA depending
on the type of the sample and the purpose from extraction (organic method (phenol ndash chloroform )-chlex ndashmethanol - commercial kit (FTA)
In genral all type of methods are following three coral steps
1-lyses cell wall by lyseis buffer
2-precipitation of proteins
3 -precipitation of DNA
1-Breaking the cells open commonly referred to as cell disruption to expose the DNA within
2-Removing membrane lipids by adding a detergent
Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents
Lysis bufferLysis buffer bull 50 mM Tris-HCI pH 80 to maintain the pH
of the solution at a level where DNA is stable 1 SDS to break open the cell and nuclear membranes allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins making them more susceptible to protease cleavage)
Precipitating the DNA with an alcohol mdash usually ethanol or isopropanol Since DNA is insoluble in these alcohols it will aggregate together giving a pellet upon centrifugation
DNA extraction ndash the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
bullSDS bullCTAB
bullProteinase K
bullFreezingbullGrinding
Chemical
Enzymatic
Mechanic
Alcohol bullEthanolbullIso-propanol
bullPhenolbullchloroform
bullSodium chloridebullSodium acetate
bullMembranebullBeads
Organic solvents
Salt
DNA binding
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Physical Characteristics of DNA DNA absorbs UV light at 260 nm Allows
quantitation DNA is water soluble DNA precipitates in alcohols DNA carries a net negative charge DNA has characteristic melting amp annealing
temperatures
Why extract DNAThe isolation of the DNA from biological sample is an essential step in the DNA technology (PCR - RFLP- cloning - hyberdization all this approaches require DNA as template
bulldisease diagnosis bullDNA sequencing
bullgenetically modified organisms (GMO) - agriculture
pharmaceutical
Nucleic Acid PreparationSample Source
Whole blood Buffy coat Serum or plasma Bone material Buccal cells Cultured cells
Amniocytes or amniotic fluid
Dried blood spots Fresh or frozen tissue
(biopsy material) Sputum urine CSF or
other body fluids Fixed or paraffin-
embedded tissue
How to isolate DNAThey are many types of methods to isolate DNA depending
on the type of the sample and the purpose from extraction (organic method (phenol ndash chloroform )-chlex ndashmethanol - commercial kit (FTA)
In genral all type of methods are following three coral steps
1-lyses cell wall by lyseis buffer
2-precipitation of proteins
3 -precipitation of DNA
1-Breaking the cells open commonly referred to as cell disruption to expose the DNA within
2-Removing membrane lipids by adding a detergent
Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents
Lysis bufferLysis buffer bull 50 mM Tris-HCI pH 80 to maintain the pH
of the solution at a level where DNA is stable 1 SDS to break open the cell and nuclear membranes allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins making them more susceptible to protease cleavage)
Precipitating the DNA with an alcohol mdash usually ethanol or isopropanol Since DNA is insoluble in these alcohols it will aggregate together giving a pellet upon centrifugation
DNA extraction ndash the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
bullSDS bullCTAB
bullProteinase K
bullFreezingbullGrinding
Chemical
Enzymatic
Mechanic
Alcohol bullEthanolbullIso-propanol
bullPhenolbullchloroform
bullSodium chloridebullSodium acetate
bullMembranebullBeads
Organic solvents
Salt
DNA binding
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Why extract DNAThe isolation of the DNA from biological sample is an essential step in the DNA technology (PCR - RFLP- cloning - hyberdization all this approaches require DNA as template
bulldisease diagnosis bullDNA sequencing
bullgenetically modified organisms (GMO) - agriculture
pharmaceutical
Nucleic Acid PreparationSample Source
Whole blood Buffy coat Serum or plasma Bone material Buccal cells Cultured cells
Amniocytes or amniotic fluid
Dried blood spots Fresh or frozen tissue
(biopsy material) Sputum urine CSF or
other body fluids Fixed or paraffin-
embedded tissue
How to isolate DNAThey are many types of methods to isolate DNA depending
on the type of the sample and the purpose from extraction (organic method (phenol ndash chloroform )-chlex ndashmethanol - commercial kit (FTA)
In genral all type of methods are following three coral steps
1-lyses cell wall by lyseis buffer
2-precipitation of proteins
3 -precipitation of DNA
1-Breaking the cells open commonly referred to as cell disruption to expose the DNA within
2-Removing membrane lipids by adding a detergent
Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents
Lysis bufferLysis buffer bull 50 mM Tris-HCI pH 80 to maintain the pH
of the solution at a level where DNA is stable 1 SDS to break open the cell and nuclear membranes allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins making them more susceptible to protease cleavage)
Precipitating the DNA with an alcohol mdash usually ethanol or isopropanol Since DNA is insoluble in these alcohols it will aggregate together giving a pellet upon centrifugation
DNA extraction ndash the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
bullSDS bullCTAB
bullProteinase K
bullFreezingbullGrinding
Chemical
Enzymatic
Mechanic
Alcohol bullEthanolbullIso-propanol
bullPhenolbullchloroform
bullSodium chloridebullSodium acetate
bullMembranebullBeads
Organic solvents
Salt
DNA binding
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Nucleic Acid PreparationSample Source
Whole blood Buffy coat Serum or plasma Bone material Buccal cells Cultured cells
Amniocytes or amniotic fluid
Dried blood spots Fresh or frozen tissue
(biopsy material) Sputum urine CSF or
other body fluids Fixed or paraffin-
embedded tissue
How to isolate DNAThey are many types of methods to isolate DNA depending
on the type of the sample and the purpose from extraction (organic method (phenol ndash chloroform )-chlex ndashmethanol - commercial kit (FTA)
In genral all type of methods are following three coral steps
1-lyses cell wall by lyseis buffer
2-precipitation of proteins
3 -precipitation of DNA
1-Breaking the cells open commonly referred to as cell disruption to expose the DNA within
2-Removing membrane lipids by adding a detergent
Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents
Lysis bufferLysis buffer bull 50 mM Tris-HCI pH 80 to maintain the pH
of the solution at a level where DNA is stable 1 SDS to break open the cell and nuclear membranes allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins making them more susceptible to protease cleavage)
Precipitating the DNA with an alcohol mdash usually ethanol or isopropanol Since DNA is insoluble in these alcohols it will aggregate together giving a pellet upon centrifugation
DNA extraction ndash the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
bullSDS bullCTAB
bullProteinase K
bullFreezingbullGrinding
Chemical
Enzymatic
Mechanic
Alcohol bullEthanolbullIso-propanol
bullPhenolbullchloroform
bullSodium chloridebullSodium acetate
bullMembranebullBeads
Organic solvents
Salt
DNA binding
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
How to isolate DNAThey are many types of methods to isolate DNA depending
on the type of the sample and the purpose from extraction (organic method (phenol ndash chloroform )-chlex ndashmethanol - commercial kit (FTA)
In genral all type of methods are following three coral steps
1-lyses cell wall by lyseis buffer
2-precipitation of proteins
3 -precipitation of DNA
1-Breaking the cells open commonly referred to as cell disruption to expose the DNA within
2-Removing membrane lipids by adding a detergent
Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents
Lysis bufferLysis buffer bull 50 mM Tris-HCI pH 80 to maintain the pH
of the solution at a level where DNA is stable 1 SDS to break open the cell and nuclear membranes allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins making them more susceptible to protease cleavage)
Precipitating the DNA with an alcohol mdash usually ethanol or isopropanol Since DNA is insoluble in these alcohols it will aggregate together giving a pellet upon centrifugation
DNA extraction ndash the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
bullSDS bullCTAB
bullProteinase K
bullFreezingbullGrinding
Chemical
Enzymatic
Mechanic
Alcohol bullEthanolbullIso-propanol
bullPhenolbullchloroform
bullSodium chloridebullSodium acetate
bullMembranebullBeads
Organic solvents
Salt
DNA binding
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
1-Breaking the cells open commonly referred to as cell disruption to expose the DNA within
2-Removing membrane lipids by adding a detergent
Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents
Lysis bufferLysis buffer bull 50 mM Tris-HCI pH 80 to maintain the pH
of the solution at a level where DNA is stable 1 SDS to break open the cell and nuclear membranes allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins making them more susceptible to protease cleavage)
Precipitating the DNA with an alcohol mdash usually ethanol or isopropanol Since DNA is insoluble in these alcohols it will aggregate together giving a pellet upon centrifugation
DNA extraction ndash the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
bullSDS bullCTAB
bullProteinase K
bullFreezingbullGrinding
Chemical
Enzymatic
Mechanic
Alcohol bullEthanolbullIso-propanol
bullPhenolbullchloroform
bullSodium chloridebullSodium acetate
bullMembranebullBeads
Organic solvents
Salt
DNA binding
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Add Lysis buffer to cells to break open cell and nuclear membranes and release nuclear contents
Lysis bufferLysis buffer bull 50 mM Tris-HCI pH 80 to maintain the pH
of the solution at a level where DNA is stable 1 SDS to break open the cell and nuclear membranes allowing the DNA to be released into the solution (SDS also denatures and unfolds proteins making them more susceptible to protease cleavage)
Precipitating the DNA with an alcohol mdash usually ethanol or isopropanol Since DNA is insoluble in these alcohols it will aggregate together giving a pellet upon centrifugation
DNA extraction ndash the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
bullSDS bullCTAB
bullProteinase K
bullFreezingbullGrinding
Chemical
Enzymatic
Mechanic
Alcohol bullEthanolbullIso-propanol
bullPhenolbullchloroform
bullSodium chloridebullSodium acetate
bullMembranebullBeads
Organic solvents
Salt
DNA binding
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Precipitating the DNA with an alcohol mdash usually ethanol or isopropanol Since DNA is insoluble in these alcohols it will aggregate together giving a pellet upon centrifugation
DNA extraction ndash the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
bullSDS bullCTAB
bullProteinase K
bullFreezingbullGrinding
Chemical
Enzymatic
Mechanic
Alcohol bullEthanolbullIso-propanol
bullPhenolbullchloroform
bullSodium chloridebullSodium acetate
bullMembranebullBeads
Organic solvents
Salt
DNA binding
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
DNA extraction ndash the basic concept
Cell lysis
Protein removal
DNA precipitation
Process Common procedure
bullSDS bullCTAB
bullProteinase K
bullFreezingbullGrinding
Chemical
Enzymatic
Mechanic
Alcohol bullEthanolbullIso-propanol
bullPhenolbullchloroform
bullSodium chloridebullSodium acetate
bullMembranebullBeads
Organic solvents
Salt
DNA binding
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
HOW
Extract
Cells
Pure DNA
Organic extraction
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
DNA Isolation MethodsLiquid Phase Organic Extraction
Phenol chloroformisoamyl alcohol Since phenol and water are immiscible two phases form -
a water phase and a phenol phase The phases are then mixed thoroughly This forces the
phenol into the water layer where it forms an emulsion of droplets throughout The proteins in the water phase are denatured and partition into the phenol while the DNA stays in the water
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
The mixture is then centrifuged and the phases separate The DNA-containing water phase can now be pipetted off and the phenolprotein solution is discarded
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Ethanol precipitation
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Time-consuming
Hazardous organic solvents
Residual amounts of organic solvents interfere
Disadvantages
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
DNA extraction kit 1048708 Two main advantages Saves time and makes the process of DNA purification a
relatively easy and straightforward process Can handle up to 100 μg of DNA
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Chelex Extraction Method
bull More rapid than organic extraction method bull Involves few steps and fewer opportunities
for contamination
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Chelex extraction
1 Put sample in tube
2 Add 5 Chelexreg beads vortex
3 Boil at 100degC
Supernatant can be used directly for quantitationPCR
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Evaluation of Nucleic Acids
Spectrophotometrically bull quantity bull quality
Assessment of DNA quality
bull gel electrophoresis
Assessment of DNA quality
bull gel electrophoresis
