What reagent can replace isoamyl alcohol in DNA extraction, when used in combination with chloroform?
3-Methylbutanol
ACS Reagent
Product Number 32,002-1
Store at Room Temperature
Exact replacement for Product Number I 0640
Product Description
Molecular Formula: C5
H12O
Molecular Weight: 88.15
CAS Number: 123-51-3
Synonyms: isoamyl alcohol, isopentyl alcohol,
3-methyl-1-butanol
This product is designated as ACS Reagent grade and
meets the specifications of the American Chemical
Society (ACS) for reagent chemicals.
3-Methyl-1-butanol, commonly called isoamyl alcohol,
is routinely used in molecular biology, notably in the
purification of DNA. It is widely used in conjunction
with phenol and chloroform, for the removal of proteins
from the nucleic acid solutions by extraction. The
addition of chloroform and isoamyl alcohol in the
extraction protocol deals with two issues that the use
of phenol alone does not completely address:
1. Isoamyl alcohol helps to inhibit RNase activity,
which phenol does not completely inhibit.
2. Isoamyl alcohol helps to prevent the solubilization
in the phenol phase of long RNA molecules with
long poly(A) portions.
In addition, isoamyl alcohol reduces foaming during
the extraction process. Isoamyl alcohol may also be
used in the extraction of ethidium bromide from DNA
solutions and in the radiolabeling of RNA transcripts in
nuclei that have been isolated from tissue.1
Isoamyl alcohol is often used in the HPLC analysis of
various pharmaceuticals and metabolites.2,3,4 It has
been used in the analysis of oxidized and reduced
pyridine nucleotides and adenylates in organic phenol
extracts from mitochondria.5
A phenol:chloroform:
isoamyl alcohol procedure for the extraction of
chloroplast DNA that avoids density gradient
differential centrifugation has been published.6
A
protocol for the isolation of mRNA from a thermophilic
cyanobacterium that incorporates a
phenol:chloroform:isoamyl alcohol mixture has been
described.7
Precautions and Disclaimer
For Laboratory Use Only. Not for drug, household or
other uses.
Preparation Instructions
This product is miscible in ethanol [0.1 ml/ml,
10% (v/v)], yielding a clear, colorless solution.
References
1. Molecular Cloning: A Laboratory Manual, 3rd ed.,
Sambrook, J. and Russell, D.W., CSHL Press
(Cold Spring Harbor, NY: 2001), pp. 1.73,
6.24-6.27, 17.27-17.28, A1.23, A8.10.
2. van de Merbel, N. C., et al., Validated liquid
chromatographic method for the determination of
bexarotene in human plasma. J. Chromatogr. B
Analyt. Technol. Biomed. Life Sci., 775(2),
189-195 (2002).
3. Dawson, M., et al., A rapid and sensitive highperformance liquid chromatography-electrospray
ionization-triple quadrupole mass spectrometry
method for the quantitation of oxycodone in
human plasma. J. Chromatogr. Sci., 40(1), 40-44
(2002).
4. De Baere, S. M., et al., Identification and
quantitation of despropionyl-bezitramide in
postmortem samples by liquid chromatography
coupled to electrospray ionization tandem mass
spectrometry. Anal. Chem., 71(14), 2908-2914
(1999). 5. Noack, H., et al., Evaluation of a procedure for the
simultaneous determination of oxidized and
reduced pyridine nucleotides and adenylates in
organic phenol extracts from mitochondria. Anal.
Biochem., 202(1), 162-165 (1992).
6. Mariac, C., et al., Chloroplast DNA extraction from
herbaceous and woody plants for direct restriction
fragment length polymorphism analysis.
Biotechniques, 28(1), 110-113 (2000).
7. Luo, X. Z., et al., Isolation of full-length RNA from
a thermophilic cyanobacterium. Biotechniques,
23(5), 904-906, 908, 910 (1997).
GCY/NSB 11/03
Sigma brand products are sold through Sigma-Ald
why we use isoamyl ...then see which chemial has the same effect of isoamyl then you can replace but take care the effect on DNA ,NOTE that ,should be an alcoholic in nature to be easy for DNA preciptation
Hi Suchi
I also agree with Nadine....there is no need to replace isoamyl alcohol...the procedureis very well established and also does not take much time.....there is not much advantage in this replacement
bye
Hi Nadine and Ajay.. isoamyl alcohol is presently unavailable in my lab and i have to initiate the procedure early. I have to use 49:1 ratio of chloroform and isoamyl alcohol, so can i replace it by some other reagent?
Mohamed, it is used to prevent the foaming caused by phenol during extraction but my procedure doesnt involve the use of phenol. So, can I replace it by other reagent?
Hi Suchi
The easieist solution will be to borrow from some neighbouring lab...since you need it in very small amount....It is also very likely that any replacement for isoamyl alcohol may not be available in your lab...and you'll also have to spend some time in optimizing the procedure....best will be to borrow it from a friend till your lab gets the supply
bye
Chloroform isoamyl alcohol is a type of detergent. It binds to protein and lipids of cell membrane and dissolve them. By this it will disrupt the bonds that hold the cell membrane together and cause it to breakdown. It then form complexes with these lipids and proteins, causing them to precipitate out of solution.
Note that lipids and proteins are non-aqueous compound and DNA/RNA are aqueous compound. The detergent binds to non-aqueous compound.
o separate two layer.DNA is insoluble in alcohol so it will floats up and all other cell components will precipitate at the bottom of the test tube.CIA also helps to isolate the CTAB solution so it will be easy to obtain the DNA.
Some people use pure chlorophorm instead of chloroform:isoamyl alcohol
Phenolic DNA Purification ---
Background and Protocol
(acc. to Sambrook and Russell, Molecular Cloning, Third Edition, Cold Spring Harbour Laboratory Press, Cold
Spring Harbour, New York, 2001)
BACKGROUND
In molecular cloning, one of the most basic procedures is the purification of nuclear acids. During this
process, it is often necessary to inactivate and/or remove enzymes or other proteins that derive from cell
extracts or that were needed during a step of cloning procedure. This removal of proteins can often be
carried out by simply extracting the aqueous solution of nucleic acids with phenol, phenol/chloroform or
phenol/chloroform/isoamyl alcohol.
The most standardised version of this process is to first extract with phenol/chloroform (1:1), optionally
containing hydroxyquinoiine at 0.1 %, followed by one or two purification steps with chloroform. This
additional chloroform extraction removes any residual phenol from the solution containing the nucleic
acids. For a detailed protocol, see below.
Note: Deproteinisation is more efficient when two different organic solvents are used instead of one
during the process.
In 1956, Kirby published the first use of phenol to purify nucleic acids [1]. His idea based on the
observation of Grassmann and Deffner (1953), who had previously reported the potency of phenol to
extract proteins from aqueous solutions [2]. In his paper, Kirby showed that extraction of homogenates
of mammalian tissue with a two-phase phenol-H2
O mixture at room temperature led to partitioning of
RNA into the aqueous layer. DNA remained associated with the proteins in the interphase. Kirby then
showed that after replacement of pure water by solutions of anionic salts both molecules, RNA as well
as DNA, could be found in the aqueous phase. Subsequently, anionic salts in their function as liberators
of nucleic acids from proteins were replaced by strong anionic detergents like SDS. The basic approach
published in this primary paper, however, was never profoundly altered and still forms the basis of many
purification methods presently in use.
Note: The function of phenol during this process is most probably the same as that of a protein
solvent – extraction of proteins that have been dissociated from nucleic acid by detergents.
3-Methylbutanol
ACS Reagent
Product Number 32,002-1
Store at Room Temperature
Exact replacement for Product Number I 0640
Product Description
Molecular Formula: C5
H12O
Molecular Weight: 88.15
CAS Number: 123-51-3
Synonyms: isoamyl alcohol, isopentyl alcohol,
3-methyl-1-butanol
This product is designated as ACS Reagent grade and
meets the specifications of the American Chemical
Society (ACS) for reagent chemicals.
3-Methyl-1-butanol, commonly called isoamyl alcohol,
is routinely used in molecular biology, notably in the
purification of DNA. It is widely used in conjunction
with phenol and chloroform, for the removal of proteins
from the nucleic acid solutions by extraction. The
addition of chloroform and isoamyl alcohol in the
extraction protocol deals with two issues that the use
of phenol alone does not completely address:
1. Isoamyl alcohol helps to inhibit RNase activity,
which phenol does not completely inhibit.
2. Isoamyl alcohol helps to prevent the solubilization
in the phenol phase of long RNA molecules with
long poly(A) portions.
In addition, isoamyl alcohol reduces foaming during
the extraction process. Isoamyl alcohol may also be
used in the extraction of ethidium bromide from DNA
solutions and in the radiolabeling of RNA transcripts in
nuclei that have been isolated from tissue.1
Isoamyl alcohol is often used in the HPLC analysis of
various pharmaceuticals and metabolites.2,3,4 It has
been used in the analysis of oxidized and reduced
pyridine nucleotides and adenylates in organic phenol
extracts from mitochondria.5
A phenol:chloroform:
isoamyl alcohol procedure for the extraction of
chloroplast DNA that avoids density gradient
differential centrifugation has been published.6
A
protocol for the isolation of mRNA from a thermophilic
cyanobacterium that incorporates a
phenol:chloroform:isoamyl alcohol mixture has been
described.7
Precautions and Disclaimer
For Laboratory Use Only. Not for drug, household or
other uses.
Preparation Instructions
This product is miscible in ethanol [0.1 ml/ml,
10% (v/v)], yielding a clear, colorless solution.
References
1. Molecular Cloning: A Laboratory Manual, 3rd ed.,
Sambrook, J. and Russell, D.W., CSHL Press
(Cold Spring Harbor, NY: 2001), pp. 1.73,
6.24-6.27, 17.27-17.28, A1.23, A8.10.
2. van de Merbel, N. C., et al., Validated liquid
chromatographic method for the determination of
bexarotene in human plasma. J. Chromatogr. B
Analyt. Technol. Biomed. Life Sci., 775(2),
189-195 (2002).
3. Dawson, M., et al., A rapid and sensitive highperformance liquid chromatography-electrospray
ionization-triple quadrupole mass spectrometry
method for the quantitation of oxycodone in
human plasma. J. Chromatogr. Sci., 40(1), 40-44
(2002).
4. De Baere, S. M., et al., Identification and
quantitation of despropionyl-bezitramide in
postmortem samples by liquid chromatography
coupled to electrospray ionization tandem mass
spectrometry. Anal. Chem., 71(14), 2908-2914
(1999). 5. Noack, H., et al., Evaluation of a procedure for the
simultaneous determination of oxidized and
reduced pyridine nucleotides and adenylates in
organic phenol extracts from mitochondria. Anal.
Biochem., 202(1), 162-165 (1992).
6. Mariac, C., et al., Chloroplast DNA extraction from
herbaceous and woody plants for direct restriction
fragment length polymorphism analysis.
Biotechniques, 28(1), 110-113 (2000).
7. Luo, X. Z., et al., Isolation of full-length RNA from
a thermophilic cyanobacterium. Biotechniques,
23(5), 904-906, 908, 910 (1997).
GCY/NSB 11/03
Sigma brand products are sold through Sigma-Ald
@ Suchi, please can I know which of the options you use. I am actually faced with similar scenario like yours, and I am opting to use only chloroform as one of Mohamed suggestions.
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