Wednesday, November 08, 2006
ETHANOL PRECIPITATION OF NUCLEIC ACIDS
To ethanol precipitate nucleic acids, add salt, mix, add ethanol, mix, then freeze. Centrifuge to pellet the material.
Choice of salts:
Sodium acetate - estimate aqueous volume of DNA
Add 1/10 of this volume from a 3M sodium acetate stock
Ammonium acetate - estimate aqueous volume of DNA
Add 1/2 of this volume from a 7.5 M ammonium acetate stock.
(Final about 2.5 M, much higher than sodium cation)
""Ammonium ions help keep free nucleotides in solution.""
當你要label DNA 時, 不需用commercial column, 就可以去除free nucleotide
(請注意:如果DNA要進行Kinase反應， 則不要用ammonium acetate; 如果sample 內有SDS,建議用NaCl or NaOAc, 因為SDS 較不會沉澱)
Ethanol:
Use absolute ethanol (i.e. 200 proof = 100%) only.
Add 2.5 - 3 times the volume of the DNA/salt mixture. Mix well before freezing.
Freezing Conditions:
Freeze in a dry ice /ethanol bath for 20 minutes, a -70°C freezer for a minimum of 40 minutes, or a -20°C freezer overnight. The latter must be used for precipitating DNA in concentrated CsCl solutions since the CsCl salt will fall out of solution at lower temperatures.
RECIPES
3M Sodium Acetate, pH 5.5
18.46 g sodium acetate
Adjust pH with glacial acetic acid (>8 ml), QS to 75 ml with water and autoclave.
7.5 M Ammonium Acetate (mw=77.08)
57.81 g ammonium acetate per 100 ml solution
Dissolve 57.81 g ammonium acetate in about 45ml autoclaved water in a sterile container. QS to 100 ml, filter sterilize with Nalgene 0.45 or 0.2 micron filter. DO NOT HEAT or AUTOCLAVE!
Send comments and updates to Dr. Bart Frank, Arthritis and Immunology Program, OMRF
=====================================================================================
another reference for DNA precipitation:
Concentrating Nucleic Acids
The need to concentrate DNA or RNA from dilute, aqueous solution arises quite frequently, for example, in almost all protocols for purification of nucleic acids from cells. The method most commonly used for this purpose is precipitation with ethanol or isopropanol.
Precipitation of DNA
A routine protocol for precipitating DNA entails the following simple steps:
1. Adjust the concentration of monovalent cations in the sample by adding 1/10th volume of 3 M sodium acetate to the sample.
2. Add 2 volumes of ethanol and mix gently. If there is sufficient DNA in the sample, you will see a white precipitate form very rapidly.
3. Recover the DNA by centrifugation, and rinse the resulting pellet with 70% ethanol to remove residual salt.
4. Evaporate off residual ethanol and resuspend the DNA in the desired buffer to the desired concentration.
The image to the right shows samples of genomic DNA before and after addition of sodium acetate and ethanol. The precipitate became visible within a few seconds of adding ethanol. When the DNA in contained in small volumes, the procedures is usually carried out in 1.5 ml microcentrifuge tubes.
Like many procedures in molecular biology, there are several effective variations-on-a-theme for preciptating DNA from solution, and with a few exceptions, the choice of which to use is largely a matter of personal preference. The significant variables for nucleic acid precipitation are:
* Type and concentration of monovalent cation: The frequently-used sources of cations are:
o Sodium acetate at a final concentration of 0.3 M (3 M stock solution).
o Sodium chloride at a final concentration of 0.2 M (5 M stock solution).
o Ammonium acetate at a final concentration of 2.5 M (7.5 M stock solution)
o Lithium chloride at a final concentration of 0.8 M (8 M stock solution).
In some situations, one salt is preferred over another. For example, ammonium acetate should not be used if the DNA is going to be phosphorylated with polynucleotide kinase. If the DNA contains the detergent SDS, sodium chloride is the choice because it allows SDS to remain soluble in 70% ethanol.
* Ethanol versus isopropanol: Isopropanol is an effective alternative to ethanol and has the advantage of precipitating DNA at lower concentrations. Instead of mixing 2 volumes of ethanol with the DNA-salt solution, addition of one volume of isopropanol will suffice.
* Time and temperature allowed for precipitation: Precipitation occurs very rapidly except when DNA content is very low (i.e. <> 2 hours on ice, the RNAs are collected by centrifugation. This method should not be used to prepare RNA for reverse transcription
Choice of salts:
Sodium acetate - estimate aqueous volume of DNA
Add 1/10 of this volume from a 3M sodium acetate stock
Ammonium acetate - estimate aqueous volume of DNA
Add 1/2 of this volume from a 7.5 M ammonium acetate stock.
(Final about 2.5 M, much higher than sodium cation)
""Ammonium ions help keep free nucleotides in solution.""
當你要label DNA 時, 不需用commercial column, 就可以去除free nucleotide
(請注意:如果DNA要進行Kinase反應， 則不要用ammonium acetate; 如果sample 內有SDS,建議用NaCl or NaOAc, 因為SDS 較不會沉澱)
Ethanol:
Use absolute ethanol (i.e. 200 proof = 100%) only.
Add 2.5 - 3 times the volume of the DNA/salt mixture. Mix well before freezing.
Freezing Conditions:
Freeze in a dry ice /ethanol bath for 20 minutes, a -70°C freezer for a minimum of 40 minutes, or a -20°C freezer overnight. The latter must be used for precipitating DNA in concentrated CsCl solutions since the CsCl salt will fall out of solution at lower temperatures.
RECIPES
3M Sodium Acetate, pH 5.5
18.46 g sodium acetate
Adjust pH with glacial acetic acid (>8 ml), QS to 75 ml with water and autoclave.
7.5 M Ammonium Acetate (mw=77.08)
57.81 g ammonium acetate per 100 ml solution
Dissolve 57.81 g ammonium acetate in about 45ml autoclaved water in a sterile container. QS to 100 ml, filter sterilize with Nalgene 0.45 or 0.2 micron filter. DO NOT HEAT or AUTOCLAVE!
Send comments and updates to Dr. Bart Frank, Arthritis and Immunology Program, OMRF
=====================================================================================
another reference for DNA precipitation:
Concentrating Nucleic Acids
The need to concentrate DNA or RNA from dilute, aqueous solution arises quite frequently, for example, in almost all protocols for purification of nucleic acids from cells. The method most commonly used for this purpose is precipitation with ethanol or isopropanol.
Precipitation of DNA
A routine protocol for precipitating DNA entails the following simple steps:
1. Adjust the concentration of monovalent cations in the sample by adding 1/10th volume of 3 M sodium acetate to the sample.
2. Add 2 volumes of ethanol and mix gently. If there is sufficient DNA in the sample, you will see a white precipitate form very rapidly.
3. Recover the DNA by centrifugation, and rinse the resulting pellet with 70% ethanol to remove residual salt.
4. Evaporate off residual ethanol and resuspend the DNA in the desired buffer to the desired concentration.
The image to the right shows samples of genomic DNA before and after addition of sodium acetate and ethanol. The precipitate became visible within a few seconds of adding ethanol. When the DNA in contained in small volumes, the procedures is usually carried out in 1.5 ml microcentrifuge tubes.
Like many procedures in molecular biology, there are several effective variations-on-a-theme for preciptating DNA from solution, and with a few exceptions, the choice of which to use is largely a matter of personal preference. The significant variables for nucleic acid precipitation are:
* Type and concentration of monovalent cation: The frequently-used sources of cations are:
o Sodium acetate at a final concentration of 0.3 M (3 M stock solution).
o Sodium chloride at a final concentration of 0.2 M (5 M stock solution).
o Ammonium acetate at a final concentration of 2.5 M (7.5 M stock solution)
o Lithium chloride at a final concentration of 0.8 M (8 M stock solution).
In some situations, one salt is preferred over another. For example, ammonium acetate should not be used if the DNA is going to be phosphorylated with polynucleotide kinase. If the DNA contains the detergent SDS, sodium chloride is the choice because it allows SDS to remain soluble in 70% ethanol.
* Ethanol versus isopropanol: Isopropanol is an effective alternative to ethanol and has the advantage of precipitating DNA at lower concentrations. Instead of mixing 2 volumes of ethanol with the DNA-salt solution, addition of one volume of isopropanol will suffice.
* Time and temperature allowed for precipitation: Precipitation occurs very rapidly except when DNA content is very low (i.e. <> 2 hours on ice, the RNAs are collected by centrifugation. This method should not be used to prepare RNA for reverse transcription
# posted by yuanchin : 6:04 AM
