What is the Function of a Tris Buffer in DNA Extraction water-soluble solid.

Its conjugate base is

Tris, or tris(hydroxymethyl) aminomethane, is a common named ethylenediaminetetraacetate. It is widely
biological buffer, used throughout the DNA extraction used to dissolve scale. Its usefulness arises
process. During extraction from any number of sources, because of its role as a hexadentate ("six-
DNA is pH sensitive. During cell lysis, removal of unwanted toothed") ligand and chelating agent, i.e. its
cellular components and precipitation, tris is used to ability to "sequester" metal ions such as Ca2+
maintain a stable pH. Additionally, it plays a particularly and Fe3+. After being bound by EDTA, metal
important role in cell lysis. ions remain in solution but exhibit diminished
reactivity. EDTA is produced as several salts,
Tris as a Buffer notably disodium EDTA and calcium disodium
As pH can influence and be influenced by a number of EDTA. Coordination chemistry principles
cellular factors, maintaining a stable pH is essential to
experimental science. Biological buffers, like tris, are In coordination chemistry, EDTA4- is a member of the
important because they can maintain a stable pH despite polyamino carboxylic acid family of ligands. EDTA4- usually
influences that might otherwise shift the pH. binds to a metal cation through its two amines and four
Tris(hydroxymethyl) aminomethane, with a pKa of 8.1, is carboxylates. Many of the resulting coordination
an effective buffer between pH 7 and 9. Because of its compounds adopt octahedral geometry. Although of little
neutral range, tris is a commonly used buffer in biological consequence for its applications, these octahedral
labs. However, tris buffer is temperature sensitive and complexes are chiral. The anion [Co(edta)]− has been
should be used at the temperature at which it was resolved into enantiomers.[5] Many complexes of EDTA4-
originally pHed to avoid inaccuracy. adopt more complex structures due to (i) the formation of
an additional bond to water, i.e. seven-coordinate
Lysis of Cells complexes, or (ii) the displacement of one carboxylate arm
Lysis, or breaking open the cells, is the first step of DNA by water. Early work on the development of EDTA was
extraction. This is accomplished by a buffer containing tris undertaken by Gerold Schwarzenbach in the 1940s.[6] EDTA
and EDTA (ethylenediaminetetraacetic acid). EDTA binds forms especially strong complexes with Mn(II), Cu(II),
divalent cations such as calcium and magnesium. Since Fe(III), Pb (II) and Co(III).[7]
these ions help maintain the integrity of the cell
membrane, eliminating them with EDTA destabilizes the
membrane. Tris is the main buffering component; its chief Several features of EDTA's complexes are relevant to its
role is to maintain the pH of the buffer at a stable point, applications. First, because of its high denticity, this ligand
usually 8.0. Additionally, tris likely interacts with the LPS has a high affinity for metal cations:
(lipopolysaccharide) in the membrane, serving to
destabilize the membrane further.
[Fe(H2O)6]3+ + H4EDTA [Fe(EDTA)]- + 6 H2O + 4
H+ (Keq = 1025.1)
Tris Protects the DNA from pH Shifts
When the cells are broken apart, their DNA and contents
spill into the buffer. Additionally, RNase A (destroys RNA), Written in this way, the equilibrium quotient shows that
proteases (destroys proteins), and SDS (sodium dodecyl metal ions compete with protons for binding to EDTA.
sulfate, solubilizes the membrane fragments) are often Because metal ions are extensively enveloped by EDTA,
included. Taken together, this soup of cellular contents and their catalytic properties are often suppressed. Finally,
fragmented RNA and proteins can have a big impact on the since complexes of EDTA4- are anionic, they tend to be
pH of the solution. Because DNA is pH sensitive, it is highly soluble in water. For this reason, EDTA is able to
important for tris to buffer the soup and maintain the pH at dissolve deposits of metal oxides and carbonates.
a steady point.
Laboratory applications
DNA Percipitation
In the final stage of DNA extraction, the DNA itself is
In the laboratory, EDTA is widely used for scavenging metal
extracted from the solution. At this point, the DNA is
ions: In biochemistry and molecular biology, ion depletion
soluble in the buffer. To extract from the solution, the DNA
is commonly used to deactivate metal-dependent
is made insoluble by adding ethanol or isopropanol
enzymes, either as an assay for their reactivity or to
(isopropyl alcohol). When this is done, the DNA become
suppress damage to DNA or proteins. In analytical
obvious in solution as a white thready substance. Although
chemistry, EDTA is used in complexometric titrations and
DNA may be isolated from the remaining cellular
analysis of water hardness or as a masking agent to
components in this way, it is not "usable" when it is
sequester metal ions that would interfere with the
insoluble. After isolation, the alcohol is removed, and DNA
analyses. EDTA finds many specialized uses in the
must be returned to a biological buffer, like tris, to be used.
biomedical laboratories, such as in veterinary
ophthalmology as an anticollagenase to prevent the
Do It Yourself
worsening of corneal ulcers in animals. In tissue culture
Although extraction of DNA is commonly done in research
EDTA is used as a chelating agent that binds to calcium
labs generally using one of a number of commercially
and prevents joining of cadherins between cells,
available kits, anyone can do DNA extraction at home
preventing clumping of cells grown in liquid suspension, or
using common household items and green peas or spinach.
detaching adherent cells for passaging.In histopathology,
In this case, tris, nor any biological buffer, is not present to
EDTA can be used as a decalcifying agent making it
protect the DNA from pH shifts. However, it is a visual way
possible to cut sections using a microtome once the tissue
of helping students to connect with cellular DNA.
sample is demineralised. EDTA is also known to inhibit a
range of metallopeptidases, the method of inhibition
occurs via the chelation of the metal ion required for
Ethylenediaminetetraacetic acid, widely abbreviated as catalytic activity.[19].
EDTA (for other names, see Table) is a
polyamino carboxylic acid and a colourless,
Sodium lauryl sulfate (SLS), sodium laurilsulfate or associated with typical amines, e.g. condensations with
sodium dodecyl sulfate (SDS or NaDS) (C12H25SO4Na) is aldehydes.
an anionic surfactant used in many cleaning and hygiene
products. The molecule has a tail of 12 carbon atoms,
attached to a sulfate group, giving the molecule the Buffering features
amphiphilic properties required of a detergent.
Tris has a pKa of 8.06, which implies that the buffer has an
SLS is a highly effective surfactant and is used in any task effective pH range between 7.0 and 9.2.
requiring the removal of oily stains and residues. For
example, it is found in higher concentrations with industrial
products including engine degreasers, floor cleaners, and [edit] Buffer details
car wash soaps. It is used in lower concentrations with
toothpastes, shampoos, and shaving foams. It is an • The pKa declines approximately 0.03 units per
important component in bubble bath formulations for its degree Celsius rise in temperature. [2][3]
thickening effect and its ability to create a lather. • Silver-containing single-junction pH electrodes
(e.g., silver chloride electrode) are incompatible
SLS has not been proven to be carcinogenic when either with Tris (Ag-tris precipitation clogs the junction).
applied directly to skin or consumed.[1] It has however been Double-junction electrodes are resistant to this
shown to irritate the skin of the face with prolonged and problem, and non-silver containing electrodes are
constant exposure (more than an hour) in young adults.[2] A immune.
clinical study found SLS toothpaste caused a higher • A common variant of tris is tris-HCl, the acid salt.
frequency of aphthous ulcers than both cocoamidopropyl When titrated to a specific pH with the
betaine or a detergent-free paste, on 30 patients with corresponding counterion (OH- for tris-HCl, H+ for
frequent occurrences of such ulcers.[3] A clinical study tris base) they are equivalent. However, the
comparing toothpastes with and without SLS found that it molecular weights are different and must be
had no significant effect on ulcer patterns.[4] correctly accounted for in order to arrive at the
expected buffer strength.
It can be used to aid in lysing cells during DNA extraction
and for unraveling proteins in SDS-PAGE. Sodium lauryl [edit] Buffer inhibition
sulfate, in science referred to as sodium dodecyl sulfate
(SDS), is commonly used in preparing proteins for
electrophoresis in the SDS-PAGE technique.[8] This • It is reported that Tris inhibits a number of
compound works by disrupting non-covalent bonds in the enzymes [4][5], and therefore, it should be used with
proteins, denaturing them, and causing the molecules to care when studying proteins.
lose their native shape (conformation). Also, anions of SDS
bind to the main peptide chain at a ratio of one SDS anion TAE buffer
for every two amino acid residues.[citation needed] This From Wikipedia, the free encyclopedia
effectively imparts a negative charge on the protein that is Jump to: navigation, search
proportional to the mass of that protein (about 1.4 g SDS/g
protein).
TAE buffer is a buffer solution containing a mixture of Tris
base, acetic acid and EDTA.
This new negative charge is significantly greater than the
original charge of that protein. The electrostatic repulsion
that is created by binding of SDS causes proteins to unfold In molecular biology it is used in agarose electrophoresis
into a rod-like shape thereby eliminating differences in typically for the separation of nucleic acids such as DNA
shape as a factor for separation in the gel. Sodium lauryl and RNA.[1] It is made up of Tris-acetate buffer, usually at
sulfate is probably the most researched anionic surfactant pH 8.0, and EDTA, which sequesters divalent cations. TAE
compound. Like all detergent surfactants (including soaps), has a lower buffer capacity than TBE and can easily
sodium lauryl sulfate removes oils from the skin, and can become exhausted, but linear, double stranded DNA runs
cause skin and eye irritation. The critical micelle faster in TAE.
concentration (CMC) in pure water at 25°C is 0.0082 M,[9]
and the aggregation number at this concentration is Recently, Brody & Kern simplified electrophoretic buffers
usually considered to be about 62.[10] The micelle ionization by substituting TBE and TAE buffers for a more efficient
fraction (α) is around 0.3 (or 30%).[11] and inexpensive conductive media in gel systems.[2]

There is evidence that surfactants such as sodium lauryl [edit] Uses

sulfate can act as a shark repellent at concentrations on
the order of 100 parts per million. However, this does not TAE (Tris-acetate-EDTA) buffer is used as both a running
meet the desired "cloud" deterrence level of 0.1 parts per buffer and in agarose gel.[3] Its use in denaturing gradient
million. [12] [13] gel electrophoresis methods for broad-range mutation
analysis has also been described. [4] TAE has been used at
Tris (also known as THAM) is an abbreviation of the various concentrations to study the mobility of DNA in
organic compound known as solution with and without sodium chloride.[5] However, high
tris(hydroxymethyl)aminomethane, with the formula concentration of sodium chloride (and many other salts) in
(HOCH2)3CNH2. Tris is extensively used in biochemistry and a DNA sample retards its mobility. This may lead to
molecular biology.[1] In biochemistry, tris is widely used as incorrect interpretations of the resulting DNA banding
a component of buffer solutions, such as in TAE and TBE pattern.
buffer, especially for solutions of nucleic acids. It is a
primary amine and thus undergoes the reactions
Compared with TBE buffer, TAE buffer offers advantages in phosphates on the DNA, neutralizing the negative
subsequent enzymatic applications for the DNA sample. charges and allowing the DNA molecules to come
For example, if a DNA sample is going to be used in a together.
cloning experiment, the step that follows its running on an
agarose gel is to ligate (covalently link) to a cloning vector 4. Any non-starchy vegetable should work. Potatoes
(most likely a plasmid). DNA sample from TAE buffer is would not work well.
suitable for this purpose, while DNA from TBE buffer is not.
Borate in the TBE buffer is a strong inhibitor for many 5. Answers will vary.
enzymes. This enzyme inhibiting property made TBE buffer
very popular in its realm for two reasons. First, a DNA 6. DNA in cells will absorb UV light. When it does it
sample run in a TBE buffer can better keep its integrity. can be damaged. If the damage occurs in a cell
The other main reason is that the purpose for many cycle gene, then growth may become uncontrolled
agarose gel electrophoreses is to analyze the size of DNA leading to skin cancer.
molecules. In particular, the kinds of DNA analyses shown
on TV and other public media are more likely run in TBE
buffer[citation needed].
QUESTIONS

SDS is strong anionic detergent. It removes the -ve ions 1. Vocabulary check. Define each of the following:
from the protein and destroys its confirmation. Because of Buffer
loss of confirmation the protein looses its structure. The
proteins fro the cel membrane get damaged and cell gets Precipitate
broken.
SDS is a detergent that is able to denature proteins. Filter
Proteins are contaminating agents in DNA isolation. They
can interfere with the final product and result in low yield. Emulsify

SDS is used to denature the proteins and facilitate the DNA 2. What is the purpose of the lysing buffer? Describe
purification process. the role of the different chemicals if possible.

Can anyone tell what the reaction mechanism of EDTA and

Dnase during DNA isolation? Because it is said that EDTA 3. Draw a picture of the negatively charged DNA and
inhibits DNase enzymes. the positively charged sodium ions. Why are the
Na+ ions necessary for the DNA molecules to come
Re: DNA isolation & EDTA? close together?
complete disruption and lysis of cells walls and plasma
membranes of cells and organelles is an absolute 4. What other vegetables could be used for this lab?
requirement for all genonic DNA isolation
procedures.Incomplete disruption results in siginificantly 5. How pure was your DNA sample? Explain.
reduced yields.Disruption generally involves use of a lysis
buffer that contains a detergent for breaking down cellular 6. Ultraviolet light has a wavelength between 10-7
membrane.Buffers containing SDS and EDTA is and 10-9 meters. Given this information and the
recomendedfor DNA isolation.EDTA chelates divalent metal information about DNA and energy absorbance in
ions to inhibit DNases and to destabilize cell membrane for this lab, why is too much sun exposure a cause of
lysis. skin cancer?

NaCl provides Na+ ions that will block negative charge

ANSWERS TO STUDENT QUESTIONS from phosphates on DNA.

Negatively charged phosphates on DNA cause molecules to

1. Buffer: A solution that will maintain a constant pH,
repel each other. The Na+ ions will form an ionic bond with
chelate metal ions and generally maintain an
the negatively charged phosphates on the DNA,
environment that will prevent disruption of the
neutralizing the negative charges and allowing the DNA
experiment.
molecules to come together.
Precipitate: The solid which is visible when a
substance becomes insoluble from a solution.
http://www.accessexcellence.org/AE/AEC/AEF/1994/dollard_
Filter: To separate by passing a mixture through a
onionDNA.php
selective membrane.
Emulsify: To physically break up lipid into smaller
as far as my knowledge goes SDS is a deterget it helps in
lipid globules.
distroying the proteins in the sample,EDTA is a chelator
which chelates the ions lik Ca+ and Mg[req by enzym to
2. To maintain an environment in which the DNA will break DNA],and chloroform being organic solvent keeps
not be degraded and to help separate the DNA the DNA away frm the water and prevts it frm getting
from other cell components. Tris maintains a dissolved.
constant pH. EDTA chelates metal ions. NaCl
provides Na+ ions that will block negative charge TRIS maintains the pH of the solution. Basically it interacts
from phosphates on DNA. with the lipopolysaccharides present on the outer
membrane which helps to permeabilize the membrane.
3. Negatively charged phosphates on DNA cause This effect is enhanced with the addition of EDTA.
molecules to repel each other. The Na+ ions will
form an ionic bond with the negatively charged
EDTA has high affinity towards divalent ions like Ca2+,
Mn2+, Mg2+ which are cofactors for many active enzymes phenol - helps in removing protein impurity from the dna ,
inside the cells. That includes nucleases which digests DNA so we can get pure dna.
molecules. Once the cell is disrupted, nuclear envelope
goes off and the nuclear content comes into contact with chloroform - prevent shearing of dna during isolation.
the cellular content which is rich in nucleases. So the
broken cell is treated with EDTA to chelate the ions so that
nucleases loose their function and that we are able to get DNA isolation is a routine procedure to collect DNA for
good yield of DNA. subsequent molecular or forensic analysis. There are three
basic and one optional steps in a DNA extraction:
ethanol change ionic potential of DNA and remove water
molecules, which help in precipitation of DNA. 1. Breaking the cells open, commonly referred to as
cell disruption or cell lysis, to expose the DNA
SDS is an anionic detergent which disrupts cell membrane within. This is commonly achieved by grinding or
and destabilizes all hydrophobic interactions holding sonicating the sample.
macromolecules in their native form.
2. Removing membrane lipids by adding a detergent.
NaoH maintains osmotic balance. 3. Removing proteins by adding a protease (optional
but almost always done).
NaCl provides Na+ions which form ionic bond with the 4. Precipitating the DNA with an alcohol — usually ice-
negatively charged phosphate of cold ethanol or isopropanol. Since DNA is insoluble
in these alcohols, it will aggregate together, giving
a pellet upon centrifugation. This step also
DNA,thus neutralizing the effect of negative ,negative removes alcohol-soluble salt.
repulsion of DNA and helps the DNA
Refinements of the technique include adding a chelating
molecules to come closer and compact to simplify our agent to sequester divalent cations such as Mg2+ and Ca2+,
process of DNA isolation... which prevents enzymes like DNAse from degrading the
DNA.
Ethanol is a dehydrating agent. In DNA it is used for the
precipitation of DNA molecule. When a molecule is to be Cellular and histone proteins bound to the DNA can be
precipitated it should be dehydrated as the water removed either by adding a protease or by having
molecules forming a film around it prevent their precipitated the proteins with sodium or ammonium
interaction. In DNA isolation the DNA molecules are acetate, or extracted them with a phenol-chloroform
dehydrated by ethanol. mixture prior to the DNA-precipitation.

If desired, the DNA can be resolubilized in a slightly

TE buffer is a commonly used buffer solution in molecular alkaline buffer or in ultra-pure water.
biology, especially in procedures involving DNA or RNA.
"TE" is derived from its components: Tris, a common pH Chloroform. It solubilizes lipids and a lot of proteins to
buffer, and EDTA, a molecule chelating cations like Mg2+. remove them from the DNA.
The purpose of TE buffer is to protect DNA or RNA from
degradation.

• For the Tris-HCl use Tris base and adjust to desired

[edit] Recipe
pH using HCl.
• TE buffer is often used to store DNA and RNA. The
A typical recipe for making 10:1 TE buffer is: EDTA in TE chelates Mg2+ and other divalent metals
ions necessary for most causes of DNA and RNA
• 10 mM Tris, bring to pH 8.0 with HCl degradation, suppressing these processes.
• 1 mM EDTA However, downstream reactions like restriction
digests, PCR, ligations, and reverse transcription
typically require Mg2+, potentially making the
TE buffer is also called as T10E1 Buffer, and read as "T ten E presence of EDTA in the reaction problematic. So,
one buffer". To make a 100 ml solution of T10E1 Buffer, 1 ml when using DNA or RNA that was suspended in TE,
of 1 M tris HCL (pH 8.0) and 0.2 ml EDTA (0.5 M) and make you should keep track of the amount of EDTA in the
up with double distilled water up to 100ml. mix to make sure there is still enough Mg2+ for
subsequent reactions to proceed successfully. Each
Based on nuclease studies from the 1980's, the pH is EDTA molecule chelates one Mg2+ ion.
usually adjusted to 7.5 for RNA and 8.0 for DNA. The • Some protocols use TE 10:0.1 with 0.1 mM EDTA to
respective DNA and RNA nucleases are supposed to be less reduce the interaction of the EDTA with
active at these pH values, but pH 8.0 can safely be used downstream applications.
for storage of both DNA and RNA. • Some people use TE buffers with different pH's for
different applications. For example, DNA is stored
EDTA further inactivates nucleases, by binding to metal at pH 8 to reduce depurination, which is acid
ions required by these enzymes. catalyzed, while RNA is stored at a slightly lower
pH (7.5) because degradation of RNA is base-
catalyzed. Most downstream reactions will not be
Diaminoethane tetraacetic acid influenced by the slightly different pH storage
conditions.
 • For dilution of primers water for injections can be spontaneously failing to generate cloneable fragments
used rather than TE. necessitate aliquoting prior to use. The phenomenon
behind these issues is not understood.

SalI[34] Streptomyces albus

Figure 5-23. Cell fractionation by differential centrifugation.
5'GTCGAC
Generally, the cellular homogenate is first filtered or
3'CAGCTG
centrifuged at relatively low speeds to remove unbroken
5'---G TCGAC---3'
cells. Then centrifugation of the homogenate at a slightly
3'---CAGCT G---5'
faster speed or for a longer duration will selectively pellet
the nucleus the largest organelle (usually 5 10 mm in
diameter). A centrifugal force of 600 g (600 times the force
of gravity) is necessary to sediment nuclei; this is PstI, is a Type II restriction endonuclease (or
generated by a typical centrifuge rotor operating at 500 restriction enzyme) from Providencia stuartii.[1][2] PstI
revolutions per minute (rpm). The undeposited material recognition and cut site are as follows:
(the supernatant) is next centrifuged at a higher speed
(15,000 g × 5 min), which deposits the mitochondria,
chloroplasts, lysosomes,and peroxisomes. A subsequent Recognition
centrifugation in the ultracentrifuge (100,000 g × 60 min) Cut Site
Sequence
results in deposition of the plasma membrane, fragments
of the endoplasmic reticulum, and large polyribosomes. A
force of 100,000 g requires about 50,000 rpm in an 5'--CTGCA
ultracentrifuge; at this speed, the rotor chamber is kept in 5'CTGCAG 3' G--3'
a high vacuum to reduce heating due to friction between 3'GACGTC 5' 3'--G
air and the spinning rotor. The recovery of ribosomal ACGTC--5'
subunits, small polyribosomes, and particles such as
complexes of enzymes requires additional centrifugation at Streptomyces caespitosus is a species of
still higher speeds. Only the cytosol the soluble aqueous actinobacteria[1]. It produces chemotherapeutic drug
portion of the cytoplasm remains undeposited after mitomycin C.
centrifugation at 300,000 g for 2 hours. ScaI[34] Streptomyces caespitosus
5'AGTACT
3'TCATGA
EcoRI (pronounced "eco R one") is an endonuclease 5'---AGT ACT---3'
enzyme isolated from strains of E. coli, and is part of the 3'---TCA TGA---5'
restriction modification system.
Well, the coldness also stop the DNA strands from breaking
apart. Remember the DNA strands are held together by
In molecular biology, it is a commonly used restriction "fragile" hydrogen bonds.
enzyme. It creates sticky ends with 5' end overhangs. The
nucleic acid sequence where the enzyme cuts is GAATTC, When you use heat to break the nuclear membrane, the
which is a palindrome as the complementary sequence is heat can also break the hydrogen bonds. So you add the
CTTAAG. cold ethanol to keep the DNA strands together, and also
DNA is non-soluble in ethanol, so it will precipitate out.
HindIII is a type II site-specific deoxyribonuclease
restriction enzyme isolated from Haemophilus influenzae There are three basic steps in a DNA extraction, the details
that cleaves the palindromic DNA sequence AAGCTT in the of which may vary depending on the type of sample and
presence of the cofactor Mg2+ via hydrolysis.[1] any substances that may interfere with the extraction and
subsequent analysis.
The cleavage of this sequence between the AA's results in
5' overhangs on the DNA called sticky ends: 1.Break open cells by grinding or sonication, and remove
membrane lipids by adding a detergent.
2.Remove cellular and histone proteins bound to the DNA,
5'-A |A G C T T-3' by adding a protease, by precipitation with sodium or
ammonium acetate, or by using a phenol-chloroform
3'-T T C G A| A-5' extraction step.

Restriction endonucleases are used as defense 3.Precipitate DNA in cold ethanol or isopropanol.
mechanisms in prokaryotic organisms in the restriction DNA is insoluble in alcohol and clings together; there by
modification system. Their primary function is to protect helping in precipitation....and .this step also removes salt.
the host genome against invasion by foreign DNA,
primarily bacteriophage DNA. There is also evidence that 4.Wash the resulting DNA pellet with alcohol
suggests the restriction enzymes may act alongside 5.Solubilize the DNA in a slightly alkaline buffer
modification enzymes as selfish elements, or may be
involved in genetic recombination and transposition.[2]
Ethanol precipitation is a commonly used technique for
BamHI is a restriction enzyme, derived from Bacillus concentrating and de-salting nucleic acid (DNA or RNA)
amyloliquefaciens. It has the recognition site (G'GATCC), preparations in aqueous solution. The basic procedure is
and leaves a sticky end.[1] One of the earlier enzymes to be that salt and ethanol are added to the aqueous solution,
used, it is popular for historical reasons, but also because which forces the nucleic acid to precipitate out of solution.
digestion leaves a GATC overhang compatible with many The precipitated nucleic acid can then be separated from
other enzymes. Persistent issues with this specific enzyme the rest of the solution by centrifugation. The pellet is
washed in cold 70% ethanol then after a further
centrifugation step the ethanol is removed, and the nucleic o Use Sodium chloride (0,2M final conc) for
acid pellet is allowed to dry before being resuspended in DNA samples containing SDS since NaCl
clean aqueous buffer. So how does this work? keeps SDS soluble in 70% ethanol so it
won’t precipitate with the DNA.
o Use Lithium Chloride (0.8M final conc) for
RNA. This is because 2.5-3 volumes of
ethanol should be used for RNA
A bit about solubility…
precipitation and LiCl is more soluble in
ethanol than NaAc so will not precipitate,
First we need to know why nucleic acids are soluble in but beware – chloride ions will inhibit
water. Water is a polar molecule – it has a partial negative protein synthesis and DNA polymerase so
charge near the oxygen atom due the unshared pairs of LiCl is no good for RNA preps for in vitro
electrons, and partial positive charges near the hydrogen translation or reverse transcription. In
atoms (see the diagram on the right). these cases, use NaAc.
o Use Ammonium acetate (2M final conc)
Because of these charges, polar molecules, like DNA or for the removal of dNTPs, but do not use
RNA, can interact electrostatically with the water for preparation of DNA for T4
molecules, allowing them to easily dissolve in water. Polar polynucleotide kinase reactions as
molecules can therefore be described as hydrophilic and ammonium ions inhibit the enzyme.
non-polar molecules, which can’t easily interact with water
molecules, are hydrophobic. Nucleic acids are hydrophilic • To increase the yield in precipitations of low
due to the negatively charged phosphate (PO3-) groups concentration or small nucleic acid pieces
along the sugar phosphate backbone. (less than 100 nucleotides)
o Add MgCl2 to a final concentration of
The role of the salt… 0.01M
o Increase the time of incubation ice before
Ok, so back to the protocol. The role of the salt in the centrifugation to 1 hour.
protocol is to neutralize the charges on the sugar
phosphate backbone. A commonly used salt is sodium If you have anything to add, please feel free to leave a
acetate. In solution, sodium acetate breaks up into Na+ comment!
and [CH3COO]-. The positively charged sodium ions
neutralize the negative charge on the PO3- groups on the
nucleic acids, making the molecule far less hydrophilic, and http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-
therefore much less soluble in water. precipitation-of-dna-and-rna-works/

the alcohol does two things, changes the ionic potential of

The role of the ethanol… the DNA and removes water molecules. this helps the DNA
to precipitate
The electrostatic attraction between the Na+ ions in
solution and the PO3- ions are dictated by Coulomb’s Law, Read more: Why use ethanol in DNA isolation | Answerbag
which is affected by the dielectric constant of the solution. http://www.answerbag.com/q_view/1114236#ixzz10ctdxBx
Water has a high dielectric constant, which makes it fairly 7
difficult for the Na+ and PO3- to come together. Ethanol on
the other hand has a much lower dielectric constant, Ethanol has a lower dielectric constant than water (it is less
making it much easier for Na+ to interact with the PO3-, electronegative by a fair margin), so when mixed with a
shield it’s charge and make the nucleic acid less pure water/ DNA sample, it effectively lowers the dielectric
hydrophilic, causing it to drop out of solution. constant of the solution. When monovalent cations are also
added to the solution, the hydration shell that is keeping
The role of temperature… DNA dissolved (water molecules bonded to and
surrounding the dissolved DNA) is replaced by ionic (salt
bonds) and the DNA molecule is knocked out of solution
Incubation of the nucleic acid/salt/ethanol mixture at low (precipitates).
temperatures (e.g. -20 or -80C) is commonly cited in
protocols as necessary in protocols. However, according to This is how ethanol (working with cations) works to
Maniatis et al (Molecular Cloning, A Laboratory Manual 2nd precipitate DNA.
Edition… 2nd edition?? – I need to get a newer version!),
this is not required, as nucleic acids at concentrations as Read more: Why use ethanol in DNA isolation | Answerbag
low as 20ng/mL will precipitate at 0-4C so incubation for http://www.answerbag.com/q_view/1114236#ixzz10ctnh1z
15-30 minutes on ice is sufficient. Y

The wash step with 70% ethanol…

Ophiopogon japonicus (Mondo grass, Fountain plant,
This step is to wash any residual salt away from the monkey grass; Japanese: リュウノヒゲ ryu-no-
pelleted DNA. hige ("dragon's beard") or ジャノヒゲ ja-no-hige
("snake's beard")) is a species of Ophiopogon
A few tips on nucleic acid precipitation… native to Japan.

It is an evergreen, sod-forming perennial plant. The leaves

• Choice of salt are linear, 20–40 cm long. The flowers are white to pale
o Use Sodium acetate (0.3M final conc, pH
5.2) for routine DNA precipitations
lilac, borne in a short raceme on a 5–10 cm stem. The fruit Hope this helps,
is a blue berry 5 mm diameter.[1] Burr
I am assuming you are extracting DNA and it wants you to
[edit] Medicinal uses pellet the DNA.
One molecule of DNA is too small to see with the even the
most powerful
In Chinese medicine Ophiopogon japonicus tuber, known as microscope. In order to see it you need millions of strands
mai men dong (Chinese: 麥門冬), is the cardinal herb for yin wrapped
deficiency. According to the Chinese Herbal Medicine together. If you then spin this in a centrifuge it will form a
Materia Medica, the herb is sweet, slightly bitter and visible mass
slightly cold, enters the Heart, Lung and Stomach channels in the bottom of the tube. It will appear as a white dot in
and nourishes the yin of the Stomach, Spleen, Heart and the bottom of
Lungs and clears heat and quiets irritability. It is used for the tube, also known as a pellet.
hacking dry coughs, dry tongue and mouth and
constipation. Liriope spicata is used as a substitute.[2] Vanhoeck
A DNA pellet is a clump of DNA that has been precipitated
[edit] Characteristics from an aquaeous
solution by the addition of alcohol.
It is also grown as an ornamental plant, providing an
excellent groundcover. Several cultivars have been Ron Baker, Ph.D.
selected, including 'Albus' (white flowers), 'Compactus' and
'Kyoto Dwarf' (dwarf forms, not over 4–5 cm tall), and Do you need RNA? An RNAse treatment should get rid of
'Silver Dragon' (variegated, with white-striped leaves). It is some of the sticky-ness. Also try incubating it overnight at
often sold as a decorative plant for freshwater aquaria, but 4 C (though if you've been working on it over the weekend,
because it is not a true aquatic plant, it may flourish for a you've probably done this anyway). One of the (many)
few months and then die. While hardy to temperatures of gDNA protocols I have for plant DNA extraction has the
about -20 °C when dormant in winter outdoors in normal final resuspension in R40 buffer (40ug/ml RNAse A in 1 x
soil, when kept fully submerged it requires water TE), which you leave it in overnight at 4 C. Sometimes I like
temperatures of 18-25 °C. It grows well in full sun or partial to incubate it on a gentle rocking platform too, I don't know
shade. Propagation is from side shoots.[1][3] if that helps, but it gives me the illusion that I'm doing
something while it's just hanging around in the cold room
The purpose of TE buffer is to protect DNA or RNA from
degradation. It is a buffer for storage of DNA & RNA.
Whatever you do, do not vortex it! It will shear the DNA.
To degrade DNA requires metal ions. THe EDTA in TE binds
the metal ions and so slows down the degradation. The I also find that freezing it at -20C then thawing it on ice can
higher pH of the Tris also slws down DNA degradation. sometimes help with the resuspension, though you don't
want to do it more than necessary (see above re:
TES buffer is a solution made up of Tris, EDTA and NaCl. Its shearing).
primary purpose to reduce the acidity of a solution. It is pH
stable and is also isotonic. How long is your 70% EtOH wash? Sometimes increasing
that will help too.
TE is Tris-EDTA pH 8.0.
Tris is needed to keep the pH at 8.0, which is the value at And don't underestimate the amount of DNA you have. The
which the double strand is stable (right pH for having H- spec reading wont be accurate until the DNA is in solution -
bonds) and EDTA chelates the 2+ ions, which are needed better to resuspend it in a greater volume, find out that it's
for DNAses to cut DNA. In this way, DNAses cannot work. too dilute then re-precipitate it than to mess about with it
at an unknown concentration.
Tris buffer maintains the pH.. EDTA prevents DNAse
enzyme by acting as a bivalent cation chelator. So the Too much DNA for the TE volume or too dry as said before.
extracted DNA can be preserved perfectly using TE buffer... If too much DNA add a little more of TE and heat the
samples in a water bath at 55C for an 1h.

Tris EDTA buffer inhibits nucleases that will degrade the Are you sure that it's DNA?
DNA, by chelating cations required by the enzymes. Aside from using enough solvent to dissolve the DNA, DNA
is extremely soluble in aqueous solution. I wouldn't expect
Phosphate buffer offers no such protection against you to have any problem in dissolving DNA in an
degradation. appropriate volume of TE buffer.

uestion: What is a DNA pellet? How can one define this I agree with Astillius. Don't assume the DNA is insoluble
term because you still see the pellet. The pellet is probably
logically and scientifically? some other crap. But if you are measuring solubility by
This refers to the mass of DNA at the bottom of the absorbance, then you may have a problem (no DNA in
centrifuge tube after pellet or insoluble). You might try placing it in a sonicating
you do a DNA extraction. DNA is insoluble in ethanol, so water bath.
when you add
ethanol to a mixture containing DNA, the DNA will If you’re performing DNA/RNA precipitations, you will have
precipitate in the read Suzanne’s excellent article on which alcohol to use for
ethanol. Centrifuging will leave a 'pellet' of DNA. precipitating your precious samples (check out some useful
info in the comments for that article as well).
Its publication prompted the recall of a useful tip I learned enough ethanol is added electrical attraction between
from a post-doc many years ago, one of those ‘magic- phosphate groups and any positive ions present in solution
hands’ scientists where everything seemed to work first becomes strong enough to form stable ionic bonds and
time. This tip of his is on the removal of the 70% ethanol in precipitate DNA. This usually happens when ethanol makes
the final step of the precipitation process. around 64% of the solution. As the mechanism suggests
solution has to contain positive ions for precipitation to
The usual wisdom is to carefully drain off the ethanol and occur, usually Na+, NH4+ or Li+ play this role [1].
leave it to either air-dry or place in a speed-vac. The
problem is that air-drying can be too slow, while the speed- [edit] Practice
vac can be too fast meaning that you risk over-drying the
pellet making solubilisation more difficult. DNA is precipitated by first ensuring that the correct
concentration of positive ions is present in solution (too
So here’s the tip: After draining or pipetting off the 70% much will result in a lot of salt co-precipitating with DNA,
ethanol, simply: too little will result in incomplete DNA recovery) and then
adding two to three volumes of at least 95% ethanol. Many
protocols advise storing DNA at low temperature at this
• recap the tube
point but this has been shown to lower precipitation
• pulse the centrifuge to bring down the remaining efficiency [2][3]. The best efficiency is achieved at room
ethanol temperature but when possible degradation is taken into
• remove this liquid with a pipette and 200ul tip – account it is probably best to incubate DNA on wet ice.
you can get right alongside the pellet if visible. Optimal incubation time depends on the length and
Leave the tube open as you move to the next concentration of DNA. Smaller fragments and lower
sample. concentrations will require longer times to achieve the
same recovery. For very small lengths and low
By the time you’ve finished your series of tubes (even if n= concentrations over-night incubation is recommended. In
3), the pellets are dry always enough to add diluent such cases use of carriers like tRNA, glycogen or linear
immediately. Job done. This saves time and give you polyacrylamide can greatly improve recovery.
samples are just the right degree of “dry”, every time.
During incubation DNA and some salts will precipitate from
Any other tips for ethanol (or even isopropanol!) solution, in the next step this precipitate is collected by
precipitation would be great to hear. Meanwhile – thanks centrifugation in a microcentrifuge tube at high speeds
Michael. (~12,000g). Time and speed of centrifugation has the
biggest effect on DNA recovery rates. Again smaller
fragments and higher dilutions require longer and faster
DNA Precipitation
centrifugation. Centrifugation can be done either at room
temperature or in 4 °C or 0 °C. During centrifugation
[edit] Theory precipitated DNA has to move through ethanol solution to
the bottom of the tube, lower temperatures increase
DNA is polar due to its highly charged phosphate viscosity of the solution and larger volumes make the
backbone. This polarity, based on the principle of "like distance longer, so both those factors lower efficiency of
dissolves like", makes it soluble in water, which is also this process requiring longer centrifugation for the same
highly polar. The high polarity of water, reflected by high effect.[2][3] After centrifugation the supernatant solution is
value of its dielectric constant 80.1 (at 20 °C), means that removed, leaving a pellet of crude DNA. Whether the pellet
electrical force between any two charges in aqueous is visible depends on the amount of DNA and on its purity
solutions is highly diminished compared to force in vacuum (dirtier pellets are easier to see) or the use of co-
or air. precipitants.
This relation is reflected in Coulomb's law, which can be
used to calculate force acting on two charges q1 and q2 In the next step, 70% ethanol is added to the pellet, and it
separated by a distance r, the dielectric constant (also is gently mixed to break the pellet loose and wash it. This
called relative static permittivity) of the medium is present removes some of the salts present in the leftover
in the denominator of the equation ( is an electric supernatant and bound to DNA pellet making the final DNA
constant): cleaner. This suspension is centrifuged again to once again
pellet DNA and the supernatant solution is removed. This
At an atomic level this diminishing of force acting on step is repeated once.
charges results from water molecules forming hydration
shells around them. It makes water a very good solvent for Finally, the pellet is air-dried and the DNA is resuspended
charged compounds like salts. Electric force which in water or other desired buffer. It is important not to over-
normally holds salt crystals together by way of ionic bonds dry the pellet as it may lead to denaturation of DNA and
is weakened in the presence of water allowing ions to make it harder to resuspend.
separate from the crystal and spread through solution.
The same mechanism operates in the case of negatively Isopropanol can also be used instead of ethanol; the
charged phosphate groups on DNA backbone, even if precipitation efficiency of the isopropanol is higher making
positive ions are present in solution, the relatively weak one volume enough for precipitation. However, isopropanol
electric force prevents them from forming stable ionic is less volatile than ethanol and needs more time to air-dry
bonds with phosphates and precipitating out of solution. in the final step. The pellet might also adhere less tightly to
the tube when using isopropanol[1].
Ethanol is much less polar than water, its dielectric
constant is 24.3 (at 25 °C). This means that adding ethanol
to solution disrupts screening of charges by water. If
[edit] Protocol of sample and ethanol is added at 2-2.5 volumes of
sample.
1. Add 1/10 volume of Sodium Acetate (3 M, pH 5.2).
2. Add 2.5-3.0 X volume (calculated after addition of The choice of which solvent to use depends largely
sodium acetate) of at least 95% ethanol. on the volume of sample you need to precipitate.
3. Incubate on ice for 15 minutes. In case of small
DNA fragments or high dilutions overnight If you are precipitating small volumes of DNA, and you can
incubation gives best results, incubation below 0 fit the required amount of solvent into the sample tube,
°C does not significantly improve efficiency [2][3]. then ice cold ethanol is the preferred choice. You can chill
4. Centrifuge at > 14,000 x g for 30 minutes at room it (some people use liquid nitrogen or -80C to accelerate
temperature or 4 °C. the precipitation) and precipitate more DNA without the
5. Discard supernatant being careful not to throw out salt contamination that would occur from chilling
DNA pellet which may or may not be visible. isopropanol. Afterwards you need to wash the pellet with
6. Rinse with 70% Ethanol 70% ethanol to remove salt.
7. Centrifuge again for 15 minutes.
8. Discard supernatant and dissolve pellet in desired Isopropanol use useful for precipitations where you have a
buffer. Make sure the buffer comes into contact large sample volume (e.g. the eluate you get after using a
with the whole surface of the tube since a Qiagen plasmid Maxi Kit) because less solvent is needed,
significant portion of DNA may be deposited on the so you can fit the whole lot in the (15ml) tube. But because
walls instead of in the pellet.[1] salts are generally less soluble in isopropanol than in
ethanol, they have more of a tendancy to co-precipitate
DNA is insoluble in alcohol, so using ethanol causes the with the DNA. So to lessen the chances of salt
DNA to precipitate out. The colder the ethanol, the less precipitation, isopropanol precipitations are carried our at
soluble the DNA is.The colder the ethanol is the greater the room temperature with minimal incubation times. Once
amount of DNA that is precipitated. the DNA or RNA pellet is recovered from the isopropanol,
you’ll want to wash it with cold 70% ethanol to remove
excess salt and to exchange the isopropanol with the more
Since our most popular article of all time (“The Basics: How volatile ethanol. It is ok to chill the isopropanol precipitated
Ethanol Precipitation of DNA and RNA Works”) was sample, if you are sure that it is not excessively salty.
published, many of our readers have asked us to further
explain the difference between precipitating DNA with
ethanol vs. isopropanol and which is the better choice. So Because DNA is less soluble in isopropanol, isopropanol
today, I’ll meet the challenge and discuss the pros and allows precipitation of larger species and lower
cons of ethanol vs. isopropanol. concentrations of nucleic acids than ethanol, especially if
you incubate it cold and long. If you do this, just remember
to wash the pellet several times in 70% ethanol after
First, let’s review what we know about what is needed for pelleting, to reduce the amount of salt you carry over.
precipitation of DNA or RNA with ethanol:
So how do you choose when to use isopropanol and
1. Salt to neutralize the charge on the nucleic acid when to use ethanol?
backbone, causing the DNA to become less hydrophilic and
fall out of solution.
Use ethanol if:
2. Ice to chill the sample. Lower temperatures promote the
flocculation of the nucleic acids so they form a larger You have room to fit two volumes of ethanol to sample in
complex that readily pellets under the centrifugal forces of your tube
a microcentrifuge.
1. The sample needs to be stored for a long period of
3. A nucleic acid concentration high enough to force the time and will be chilled
DNA out of solution (if the conc is not high enough, you can 2. You need to precipitate very small pieces of DNA or
add a carrier nucleic acid or glycogen to enhance the you have a very low concentration of sample so
recovery). you want to chill it longer and colder.

4. Centrifugation to pellet the sample Use Isopropanol if:

The difference between isopropanol and ethanol is 1. You have limited in space in your tube and can fit
the solubility of DNA in each solvent. only 1 volume of sample
2. You need large molecular weight species because
incubation at room temperature for short periods
DNA is less soluble in isopropanol so it will fall out of of time will not be conducive to precipitating small
solution faster and at a lower concentration, but the species of nucleic acid
downside is that the salt will too. With ethanol, the DNA 3. You are in a hurry and want to accelerate the
needs to be at a higher concentration to flocculate but the precipitation of nucleic acids at room temperature
salt tends to stay soluble, even at cold temperatures.
What do I prefer? I use ethanol over isopropanol for most
DNA falls out of solution in 35% isopropanol and 0.5M salt. cases, but will use isopropanol if I need to make everything
Using ethanol, the final concentration needs to be around fit in one tube. My preferred protocol is 2 volumes of
75% with 0.5M salt. So for the typical precipitation ethanol and freeze at -20C for at least an hour or overnight
protocol, isopropanol is added from between 0.7-1 volumes for best results. I centrifuge the sample at full speed for 20
minutes to make sure I get everything down. I always wash
with 70% ethanol and then centrifuge for 10-15 minutes Washington Post Staff Writer
and keep my eye on the pellet when I decant everything. Thursday, August 11, 2005
You need to note or mark the side of the tube where the
pellet is expected to be and don’t let it out of your sight Scientists have completed a genetic map of the rice plant,
when decanting the ethanol! a scientific milestone that they hope will accelerate efforts
to feed the hungry by improving the world's most
If I use isopropanol, I avoid cold temperatures because of important food.
the excess salt that usually comes down with it. If I want to
increase the yields precipitated, I prefer to leave it Rice is the first crop plant whose complete genetic
incubating at room temperature longer vs. chilling the sequence, or genome, has been compiled and placed in
sample. When the DNA is pelleted, the pellet is sometimes computer data banks around the world. It will be a key tool
more difficult to see compared to the ethanol pellet. It can for researchers working on improved strains of rice and
be clear and glassy. Make sure, again, to note the side of other grains as they struggle to stay ahead of human
the tube where the pellet should be. Look for it before population growth. A paper describing the genome is being
decanting the isopropanol and 70% ethanol wash. After published today in the journal Nature, and the sequence
washing with ethanol, the pellet becomes visible and white. will be freely available to researchers worldwide.
I always make sure it doesn’t slip off the side of the tube
wall before decanting the supernatant. Allow the tube to
drain upside down for a few minutes and then air dry or Centrifuge to separate the DNA from the dissolved salts
speed vac dry (5 minutes is enough) and then resuspend in and sugars
buffer.
DNA EXTRACTION
Protocol and notes
Finally, for dry DNA pellets, heating the sample in buffer at Introduction
50-60C will help the DNA dissolve faster and won’t damage
the DNA. For RNA, heating can be used too (in water) at • This highlights a simple protocol for extracting DNA
temps around 42C. Overdried DNA and RNA will take from cereal plant leaves
longer to dissolve so make sure not to speed vac for too
long. • This protocol will produce a product that is suitable
as a template for polymerase chain reaction (PCR) and
restriction enzyme digestion
Genes, or “coding DNA,” are segments of DNA that contain
the chemical recipe that determines particular traits. • It will also contain RNA which can be removed with
Scientists now estimate that rice has about 37,500 genes, further purifications
located along threadlike, tightly coiled strands of DNA DNA Extraction
called chromosomes. Genes, however, are only about three Protocol Overview
percent of rice DNA; the rest is "noncoding" DNA. These
noncoding regions of the genome contain the information
• Fresh plant material is collected
that determines when and where genes are active – for • The plant material is mixed with extraction buffer
example, in which cell types and at what stages in growth and ground in a mortar and pestle
and development of rice. • The resulting liquid is centrifuged to separate the
solid plant material from the extraction buffer supernatant
More than $15 million was competitively awarded by NIFA,
NSF, and DOE, with more than $6.5 million of NIFA funding
• The supernatant is mixed with ethanol to
precipitate the DNA which will form a pellet at the bottom
awarded to The Institute for Genomic Research (TIGR) and
of the tube
the University of Arizona. Researchers at the University of
Arizona collaborated with Cold Spring Harbor Laboratory in • The supernatant is discarded
New York, Washington University, and the University of • The DNA pellet is washed with a diluted ethanol
Wisconsin. These groups coordinated their research with solution
privately funded U.S. groups, including Rutgers University, • The DNA pellet is dried and then dissolved in water
Monsanto, and Syngenta.
or a buffer

The finished genome sequence has already led to the Protocol for Collecting the Plant Material
discovery of specific gene functions, which may help in • Set up and label (according to species) TWO
fighting diseases and physiological stress. It may also lead
eppendorf tubes for each seedling you will be extracting
to improved rice breeding, including the production of
DNA from.
higher quality rice in less time.

• Add 600 µL of isopropanol to one tube in step 1

Rice has the smallest genome of all cereal grass crops and and leave the other tube empty.
was the first crop to be genetically sequenced. Since rice is
genetically similar to other grasses, the rice genome could
provide important genetic information about grasses like • Add 2.0 mL of extraction
wheat, barley, sorghum, and corn. buffer to the mortar.

Rice Genome Fully Mapped • Cut a 2 inch portion of leaf

from each plant and grind
in the mortar with the pestle
First DNA Map for a Crop Expected to Boost until the solution is deep green.
Modification Efforts Transfer 1.5 mL of solution to empty tube.
By Justin Gillis
• Centrifuge the eppendorf tube with the extraction
buffer + crushed leaf at high speed for 3 minutes (this will
pellet the leaf debris at the bottom of the tube)

• Remove 1 mL of the supernatant and transfer to

the tube containing 600 µL of isopropanol. DISCARD the
tube with the debris pellet!

• Invert the sample tubes (with supernatant and

isopropanol) 15 times and incubate them at room
temperature for 2 minutes. (note: DNA should precipitate
out of solution and be visible as “white and cottony”)
8. Centrifuge samples at high speed for 5 minutes to
pellet DNA. Remove supernatant and save the pellet (DNA
is in the pellet!).
9. Add 1000 µL of 70% ethanol to the DNA pellets. Mix by
inversion 15 times. Centrifuge at high speed for 3 minutes.
Remove and discard as much supernatant as possible.
Again, save the pellet!
10. Leave the tube open and let it air dry for 10-15
minutes. Add 50 µL of 1X TE to resuspend the DNA in
solution.

• The DNA solution is now ready to use.

• To keep DNA solution stable, keep it in the fridge

for short term storage and in the freezer for long term
storage.
• After extractions, it is a good practice to run the
DNA on an agarose gel to check its integrity and quality
• Mix 10 µL of DNA with 10 µL of loading buffer
• Load this mixture into a 1% agarose gel
Examining the Quality of the DNA Extracted

If the genomic DNA is extracted in a professional

Molecular Biology laboratory, it will give clear distinct band
in an electrophoresis gel