What is the Function of a Tris Buffer in DNA Extraction
Its conjugate base is
Tris, or tris(hydroxymethyl) aminomethane, is a common
biological buffer, used throughout the DNA extraction
process. During extraction from any number of sources,
DNA is pH sensitive. During cell lysis, removal of unwanted
cellular components and precipitation, tris is used to
maintain a stable pH. Additionally, it plays a particularly
important role in cell lysis.
Tris as a Buffer
As pH can influence and be influenced by a number of
cellular factors, maintaining a stable pH is essential to
experimental science. Biological buffers, like tris, are
important because they can maintain a stable pH despite
influences that might otherwise shift the pH.
Tris(hydroxymethyl) aminomethane, with a pKa of 8.1, is
an effective buffer between pH 7 and 9. Because of its
neutral range, tris is a commonly used buffer in biological
labs. However, tris buffer is temperature sensitive and
should be used at the temperature at which it was
originally pHed to avoid inaccuracy.
Lysis of Cells
Lysis, or breaking open the cells, is the first step of DNA
extraction. This is accomplished by a buffer containing tris
and EDTA (ethylenediaminetetraacetic acid). EDTA binds
divalent cations such as calcium and magnesium. Since
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
role is to maintain the pH of the buffer at a stable point,
usually 8.0. Additionally, tris likely interacts with the LPS
(lipopolysaccharide) in the membrane, serving to
destabilize the membrane further.
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),
proteases (destroys proteins), and SDS (sodium dodecyl
sulfate, solubilizes the membrane fragments) are often
included. Taken together, this soup of cellular contents and
fragmented RNA and proteins can have a big impact on the
pH of the solution. Because DNA is pH sensitive, it is
important for tris to buffer the soup and maintain the pH at
a steady point.
DNA Percipitation
In the final stage of DNA extraction, the DNA itself is
extracted from the solution. At this point, the DNA is
soluble in the buffer. To extract from the solution, the DNA
is made insoluble by adding ethanol or isopropanol
(isopropyl alcohol). When this is done, the DNA become
obvious in solution as a white thready substance. Although
DNA may be isolated from the remaining cellular
components in this way, it is not "usable" when it is
insoluble. After isolation, the alcohol is removed, and DNA
must be returned to a biological buffer, like tris, to be used.
Do It Yourself
Although extraction of DNA is commonly done in research
labs generally using one of a number of commercially
available kits, anyone can do DNA extraction at home
using common household items and green peas or spinach.
In this case, tris, nor any biological buffer, is not present to
protect the DNA from pH shifts. However, it is a visual way
of helping students to connect with cellular DNA.
Ethylenediaminetetraacetic acid, widely abbreviated as
EDTA (for other names, see Table) is a
polyamino carboxylic acid and a colourless,
water-soluble solid.
named ethylenediaminetetraacetate. It is widely
used to dissolve scale. Its usefulness arises
because of its role as a hexadentate ("six-
toothed") ligand and chelating agent, i.e. its
ability to "sequester" metal ions such as Ca2+
and Fe3+. After being bound by EDTA, metal
ions remain in solution but exhibit diminished
reactivity. EDTA is produced as several salts,
notably disodium EDTA and calcium disodium
EDTA. Coordination chemistry principles
In coordination chemistry, EDTA4- is a member of the
polyamino carboxylic acid family of ligands. EDTA4- usually
binds to a metal cation through its two amines and four
carboxylates. Many of the resulting coordination
compounds adopt octahedral geometry. Although of little
consequence for its applications, these octahedral
complexes are chiral. The anion [Co(edta)]− has been
resolved into enantiomers.[5] Many complexes of EDTA4-
adopt more complex structures due to (i) the formation of
an additional bond to water, i.e. seven-coordinate
complexes, or (ii) the displacement of one carboxylate arm
by water. Early work on the development of EDTA was
undertaken by Gerold Schwarzenbach in the 1940s.[6] EDTA
forms especially strong complexes with Mn(II), Cu(II),
Fe(III), Pb (II) and Co(III).[7]
Several features of EDTA's complexes are relevant to its
applications. First, because of its high denticity, this ligand
has a high affinity for metal cations:
[Fe(H2O)6]3+ + H4EDTA [Fe(EDTA)]- + 6 H2O + 4
H+ (Keq = 1025.1)
Written in this way, the equilibrium quotient shows that
metal ions compete with protons for binding to EDTA.
Because metal ions are extensively enveloped by EDTA,
their catalytic properties are often suppressed. Finally,
since complexes of EDTA4- are anionic, they tend to be
highly soluble in water. For this reason, EDTA is able to
dissolve deposits of metal oxides and carbonates.
Laboratory applications
In the laboratory, EDTA is widely used for scavenging metal
ions: In biochemistry and molecular biology, ion depletion
is commonly used to deactivate metal-dependent
enzymes, either as an assay for their reactivity or to
suppress damage to DNA or proteins. In analytical
chemistry, EDTA is used in complexometric titrations and
analysis of water hardness or as a masking agent to
sequester metal ions that would interfere with the
analyses. EDTA finds many specialized uses in the
biomedical laboratories, such as in veterinary
ophthalmology as an anticollagenase to prevent the
worsening of corneal ulcers in animals. In tissue culture
EDTA is used as a chelating agent that binds to calcium
and prevents joining of cadherins between cells,
preventing clumping of cells grown in liquid suspension, or
detaching adherent cells for passaging.In histopathology,
EDTA can be used as a decalcifying agent making it
possible to cut sections using a microtome once the tissue
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
catalytic activity.[19].
Sodium lauryl sulfate (SLS), sodium laurilsulfate or
sodium dodecyl sulfate (SDS or NaDS) (C12H25SO4Na) is
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
amphiphilic properties required of a detergent.
SLS is a highly effective surfactant and is used in any task
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
car wash soaps. It is used in lower concentrations with
toothpastes, shampoos, and shaving foams. It is an
important component in bubble bath formulations for its
thickening effect and its ability to create a lather.
SLS has not been proven to be carcinogenic when either
applied directly to skin or consumed.[1] It has however been
shown to irritate the skin of the face with prolonged and
constant exposure (more than an hour) in young adults.[2] A
clinical study found SLS toothpaste caused a higher
frequency of aphthous ulcers than both cocoamidopropyl
betaine or a detergent-free paste, on 30 patients with
frequent occurrences of such ulcers.[3] A clinical study
comparing toothpastes with and without SLS found that it
had no significant effect on ulcer patterns.[4]
It can be used to aid in lysing cells during DNA extraction
and for unraveling proteins in SDS-PAGE. Sodium lauryl
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
compound works by disrupting non-covalent bonds in the
proteins, denaturing them, and causing the molecules to
lose their native shape (conformation). Also, anions of SDS
bind to the main peptide chain at a ratio of one SDS anion
for every two amino acid residues.[citation needed] This
effectively imparts a negative charge on the protein that is
proportional to the mass of that protein (about 1.4 g SDS/g
protein).
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
into a rod-like shape thereby eliminating differences in
shape as a factor for separation in the gel. Sodium lauryl
sulfate is probably the most researched anionic surfactant
compound. Like all detergent surfactants (including soaps),
sodium lauryl sulfate removes oils from the skin, and can
cause skin and eye irritation. The critical micelle
concentration (CMC) in pure water at 25°C is 0.0082 M,[9]
and the aggregation number at this concentration is
usually considered to be about 62.[10] The micelle ionization
fraction (α) is around 0.3 (or 30%).[11]
There is evidence that surfactants such as sodium lauryl
sulfate can act as a shark repellent at concentrations on
the order of 100 parts per million. However, this does not
meet the desired "cloud" deterrence level of 0.1 parts per
million. [12] [13]
Tris (also known as THAM) is an abbreviation of the
organic compound known as
tris(hydroxymethyl)aminomethane, with the formula
(HOCH2)3CNH2. Tris is extensively used in biochemistry and
molecular biology.[1] In biochemistry, tris is widely used as
a component of buffer solutions, such as in TAE and TBE
buffer, especially for solutions of nucleic acids. It is a
primary amine and thus undergoes the reactions
associated with typical amines, e.g. condensations with
aldehydes.
Buffering features
Tris has a pKa of 8.06, which implies that the buffer has an
effective pH range between 7.0 and 9.2.
[edit] Buffer details
• The pKa declines approximately 0.03 units per
degree Celsius rise in temperature. [2][3]
• Silver-containing single-junction pH electrodes
(e.g., silver chloride electrode) are incompatible
with Tris (Ag-tris precipitation clogs the junction).
Double-junction electrodes are resistant to this
problem, and non-silver containing electrodes are
immune.
• A common variant of tris is tris-HCl, the acid salt.
When titrated to a specific pH with the
corresponding counterion (OH- for tris-HCl, H+ for
tris base) they are equivalent. However, the
molecular weights are different and must be
correctly accounted for in order to arrive at the
expected buffer strength.
[edit] Buffer inhibition
• It is reported that Tris inhibits a number of
enzymes [4][5], and therefore, it should be used with
care when studying proteins.
TAE buffer
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TAE buffer is a buffer solution containing a mixture of Tris
base, acetic acid and EDTA.
In molecular biology it is used in agarose electrophoresis
typically for the separation of nucleic acids such as DNA
and RNA.[1] It is made up of Tris-acetate buffer, usually at
pH 8.0, and EDTA, which sequesters divalent cations. TAE
has a lower buffer capacity than TBE and can easily
become exhausted, but linear, double stranded DNA runs
faster in TAE.
Recently, Brody & Kern simplified electrophoretic buffers
by substituting TBE and TAE buffers for a more efficient
and inexpensive conductive media in gel systems.[2]
[edit] Uses
TAE (Tris-acetate-EDTA) buffer is used as both a running
buffer and in agarose gel.[3] Its use in denaturing gradient
gel electrophoresis methods for broad-range mutation
analysis has also been described. [4] TAE has been used at
various concentrations to study the mobility of DNA in
solution with and without sodium chloride.[5] However, high
concentration of sodium chloride (and many other salts) in
a DNA sample retards its mobility. This may lead to
incorrect interpretations of the resulting DNA banding
pattern.
Compared with TBE buffer, TAE buffer offers advantages in
subsequent enzymatic applications for the DNA sample.
For example, if a DNA sample is going to be used in a
cloning experiment, the step that follows its running on an
agarose gel is to ligate (covalently link) to a cloning vector
(most likely a plasmid). DNA sample from TAE buffer is
suitable for this purpose, while DNA from TBE buffer is not.
Borate in the TBE buffer is a strong inhibitor for many
enzymes. This enzyme inhibiting property made TBE buffer
very popular in its realm for two reasons. First, a DNA
sample run in a TBE buffer can better keep its integrity.
The other main reason is that the purpose for many
agarose gel electrophoreses is to analyze the size of DNA
molecules. In particular, the kinds of DNA analyses shown
on TV and other public media are more likely run in TBE
buffer[citation needed].
SDS is strong anionic detergent. It removes the -ve ions
from the protein and destroys its confirmation. Because of
loss of confirmation the protein looses its structure. The
proteins fro the cel membrane get damaged and cell gets
broken.
SDS is a detergent that is able to denature proteins.
Proteins are contaminating agents in DNA isolation. They
can interfere with the final product and result in low yield.
SDS is used to denature the proteins and facilitate the DNA
purification process.
Can anyone tell what the reaction mechanism of EDTA and
Dnase during DNA isolation? Because it is said that EDTA
inhibits DNase enzymes.
Re: DNA isolation & EDTA?
complete disruption and lysis of cells walls and plasma
membranes of cells and organelles is an absolute
requirement for all genonic DNA isolation
procedures.Incomplete disruption results in siginificantly
reduced yields.Disruption generally involves use of a lysis
buffer that contains a detergent for breaking down cellular
membrane.Buffers containing SDS and EDTA is
recomendedfor DNA isolation.EDTA chelates divalent metal
ions to inhibit DNases and to destabilize cell membrane for
lysis.
ANSWERS TO STUDENT QUESTIONS
1. Buffer: A solution that will maintain a constant pH,
chelate metal ions and generally maintain an
environment that will prevent disruption of the
experiment.
Precipitate: The solid which is visible when a
substance becomes insoluble from a solution.
Filter: To separate by passing a mixture through a
selective membrane.
Emulsify: To physically break up lipid into smaller
lipid globules.
2. To maintain an environment in which the DNA will
not be degraded and to help separate the DNA
from other cell components. Tris maintains a
constant pH. EDTA chelates metal ions. NaCl
provides Na+ ions that will block negative charge
from phosphates on DNA.
3. Negatively charged phosphates on DNA cause
molecules to repel each other. The Na+ ions will
form an ionic bond with the negatively charged
phosphates on the DNA, neutralizing the negative
charges and allowing the DNA molecules to come
together.
4. Any non-starchy vegetable should work. Potatoes
would not work well.
5. Answers will vary.
6. DNA in cells will absorb UV light. When it does it
can be damaged. If the damage occurs in a cell
cycle gene, then growth may become uncontrolled
leading to skin cancer.
QUESTIONS
1. Vocabulary check. Define each of the following:
Buffer
Precipitate
Filter
Emulsify
2. What is the purpose of the lysing buffer? Describe
the role of the different chemicals if possible.
3. Draw a picture of the negatively charged DNA and
the positively charged sodium ions. Why are the
Na+ ions necessary for the DNA molecules to come
close together?
4. What other vegetables could be used for this lab?
5. How pure was your DNA sample? Explain.
6. Ultraviolet light has a wavelength between 10-7
and 10-9 meters. Given this information and the
information about DNA and energy absorbance in
this lab, why is too much sun exposure a cause of
skin cancer?
NaCl provides Na+ ions that will block negative charge
from phosphates on DNA.
Negatively charged phosphates on DNA cause molecules to
repel each other. The Na+ ions will form an ionic bond with
the negatively charged phosphates on the DNA,
neutralizing the negative charges and allowing the DNA
molecules to come together.
http://www.accessexcellence.org/AE/AEC/AEF/1994/dollard_
onionDNA.php
as far as my knowledge goes SDS is a deterget it helps in
distroying the proteins in the sample,EDTA is a chelator
which chelates the ions lik Ca+ and Mg[req by enzym to
break DNA],and chloroform being organic solvent keeps
the DNA away frm the water and prevts it frm getting
dissolved.
TRIS maintains the pH of the solution. Basically it interacts
with the lipopolysaccharides present on the outer
membrane which helps to permeabilize the membrane.
This effect is enhanced with the addition of EDTA.
EDTA has high affinity towards divalent ions like Ca2+,
Mn2+, Mg2+ which are cofactors for many active enzymes
inside the cells. That includes nucleases which digests DNA
molecules. Once the cell is disrupted, nuclear envelope
goes off and the nuclear content comes into contact with
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
good yield of DNA.
ethanol change ionic potential of DNA and remove water
molecules, which help in precipitation of DNA.
SDS is an anionic detergent which disrupts cell membrane
and destabilizes all hydrophobic interactions holding
macromolecules in their native form.
NaoH maintains osmotic balance.
NaCl provides Na+ions which form ionic bond with the
negatively charged phosphate of
DNA,thus neutralizing the effect of negative ,negative
repulsion of DNA and helps the DNA
molecules to come closer and compact to simplify our
process of DNA isolation...
Ethanol is a dehydrating agent. In DNA it is used for the
precipitation of DNA molecule. When a molecule is to be
precipitated it should be dehydrated as the water
molecules forming a film around it prevent their
interaction. In DNA isolation the DNA molecules are
dehydrated by ethanol.
TE buffer is a commonly used buffer solution in molecular
biology, especially in procedures involving DNA or RNA.
"TE" is derived from its components: Tris, a common pH
buffer, and EDTA, a molecule chelating cations like Mg2+.
The purpose of TE buffer is to protect DNA or RNA from
degradation.
[edit] Recipe
A typical recipe for making 10:1 TE buffer is:
• 10 mM Tris, bring to pH 8.0 with HCl
• 1 mM EDTA
TE buffer is also called as T10E1 Buffer, and read as "T ten E
one buffer". To make a 100 ml solution of T10E1 Buffer, 1 ml
of 1 M tris HCL (pH 8.0) and 0.2 ml EDTA (0.5 M) and make
up with double distilled water up to 100ml.
Based on nuclease studies from the 1980's, the pH is
usually adjusted to 7.5 for RNA and 8.0 for DNA. The
respective DNA and RNA nucleases are supposed to be less
active at these pH values, but pH 8.0 can safely be used
for storage of both DNA and RNA.
EDTA further inactivates nucleases, by binding to metal
ions required by these enzymes.
Diaminoethane tetraacetic acid
phenol - helps in removing protein impurity from the dna ,
so we can get pure dna.
chloroform - prevent shearing of dna during isolation.
DNA isolation is a routine procedure to collect DNA for
subsequent molecular or forensic analysis. There are three
basic and one optional steps in a DNA extraction:
1. Breaking the cells open, commonly referred to as
cell disruption or cell lysis, to expose the DNA
within. This is commonly achieved by grinding or
sonicating the sample.
2. Removing membrane lipids by adding a detergent.
3. Removing proteins by adding a protease (optional
but almost always done).
4. Precipitating the DNA with an alcohol — usually ice-
cold ethanol or isopropanol. Since DNA is insoluble
in these alcohols, it will aggregate together, giving
a pellet upon centrifugation. This step also
removes alcohol-soluble salt.
Refinements of the technique include adding a chelating
agent to sequester divalent cations such as Mg2+ and Ca2+,
which prevents enzymes like DNAse from degrading the
DNA.
Cellular and histone proteins bound to the DNA can be
removed either by adding a protease or by having
precipitated the proteins with sodium or ammonium
acetate, or extracted them with a phenol-chloroform
mixture prior to the DNA-precipitation.
If desired, the DNA can be resolubilized in a slightly
alkaline buffer or in ultra-pure water.
Chloroform. It solubilizes lipids and a lot of proteins to
remove them from the DNA.
• For the Tris-HCl use Tris base and adjust to desired
pH using HCl.
• TE buffer is often used to store DNA and RNA. The
EDTA in TE chelates Mg2+ and other divalent metals
ions necessary for most causes of DNA and RNA
degradation, suppressing these processes.
However, downstream reactions like restriction
digests, PCR, ligations, and reverse transcription
typically require Mg2+, potentially making the
presence of EDTA in the reaction problematic. So,
when using DNA or RNA that was suspended in TE,
you should keep track of the amount of EDTA in the
mix to make sure there is still enough Mg2+ for
subsequent reactions to proceed successfully. Each
EDTA molecule chelates one Mg2+ ion.
• Some protocols use TE 10:0.1 with 0.1 mM EDTA to
reduce the interaction of the EDTA with
downstream applications.
• Some people use TE buffers with different pH's for
different applications. For example, DNA is stored
at pH 8 to reduce depurination, which is acid
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
influenced by the slightly different pH storage
conditions.
 • For dilution of primers water for injections can be
used rather than TE.
Figure 5-23. Cell fractionation by differential centrifugation.
Generally, the cellular homogenate is first filtered or
centrifuged at relatively low speeds to remove unbroken
cells. Then centrifugation of the homogenate at a slightly
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
generated by a typical centrifuge rotor operating at 500
revolutions per minute (rpm). The undeposited material
(the supernatant) is next centrifuged at a higher speed
(15,000 g × 5 min), which deposits the mitochondria,
chloroplasts, lysosomes,and peroxisomes. A subsequent
centrifugation in the ultracentrifuge (100,000 g × 60 min)
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
ultracentrifuge; at this speed, the rotor chamber is kept in
a high vacuum to reduce heating due to friction between
air and the spinning rotor. The recovery of ribosomal
subunits, small polyribosomes, and particles such as
complexes of enzymes requires additional centrifugation at
still higher speeds. Only the cytosol the soluble aqueous
portion of the cytoplasm remains undeposited after
centrifugation at 300,000 g for 2 hours.
EcoRI (pronounced "eco R one") is an endonuclease
enzyme isolated from strains of E. coli, and is part of the
restriction modification system.
In molecular biology, it is a commonly used restriction
enzyme. It creates sticky ends with 5' end overhangs. The
nucleic acid sequence where the enzyme cuts is GAATTC,
which is a palindrome as the complementary sequence is
CTTAAG.
HindIII is a type II site-specific deoxyribonuclease
restriction enzyme isolated from Haemophilus influenzae
that cleaves the palindromic DNA sequence AAGCTT in the
presence of the cofactor Mg2+ via hydrolysis.[1]
The cleavage of this sequence between the AA's results in
5' overhangs on the DNA called sticky ends:
5'-A |A G C T T-3'
3'-T T C G A| A-5'
Restriction endonucleases are used as defense
mechanisms in prokaryotic organisms in the restriction
modification system. Their primary function is to protect
the host genome against invasion by foreign DNA,
primarily bacteriophage DNA. There is also evidence that
suggests the restriction enzymes may act alongside
modification enzymes as selfish elements, or may be
involved in genetic recombination and transposition.[2]
BamHI is a restriction enzyme, derived from Bacillus
amyloliquefaciens. It has the recognition site (G'GATCC),
and leaves a sticky end.[1] One of the earlier enzymes to be
used, it is popular for historical reasons, but also because
digestion leaves a GATC overhang compatible with many
other enzymes. Persistent issues with this specific enzyme
spontaneously failing to generate cloneable fragments
necessitate aliquoting prior to use. The phenomenon
behind these issues is not understood.
SalI[34] Streptomyces albus
5'GTCGAC
3'CAGCTG
5'---G TCGAC---3'
3'---CAGCT G---5'
PstI, is a Type II restriction endonuclease (or
restriction enzyme) from Providencia stuartii.[1][2] PstI
recognition and cut site are as follows:
Recognition
Cut Site
Sequence
5'--CTGCA
5'CTGCAG 3' G--3'
3'GACGTC 5' 3'--G
ACGTC--5'
Streptomyces caespitosus is a species of
actinobacteria[1]. It produces chemotherapeutic drug
mitomycin C.
ScaI[34] Streptomyces caespitosus
5'AGTACT
3'TCATGA
5'---AGT ACT---3'
3'---TCA TGA---5'
Well, the coldness also stop the DNA strands from breaking
apart. Remember the DNA strands are held together by
"fragile" hydrogen bonds.
When you use heat to break the nuclear membrane, the
heat can also break the hydrogen bonds. So you add the
cold ethanol to keep the DNA strands together, and also
DNA is non-soluble in ethanol, so it will precipitate out.
There are three basic steps in a DNA extraction, the details
of which may vary depending on the type of sample and
any substances that may interfere with the extraction and
subsequent analysis.
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,
by adding a protease, by precipitation with sodium or
ammonium acetate, or by using a phenol-chloroform
extraction step.
3.Precipitate DNA in cold ethanol or isopropanol.
DNA is insoluble in alcohol and clings together; there by
helping in precipitation....and .this step also removes salt.
4.Wash the resulting DNA pellet with alcohol
5.Solubilize the DNA in a slightly alkaline buffer
Ethanol precipitation is a commonly used technique for
concentrating and de-salting nucleic acid (DNA or RNA)
preparations in aqueous solution. The basic procedure is
that salt and ethanol are added to the aqueous solution,
which forces the nucleic acid to precipitate out of solution.
The precipitated nucleic acid can then be separated from
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
acid pellet is allowed to dry before being resuspended in
clean aqueous buffer. So how does this work?
A bit about solubility…
First we need to know why nucleic acids are soluble in
water. Water is a polar molecule – it has a partial negative
charge near the oxygen atom due the unshared pairs of
electrons, and partial positive charges near the hydrogen
atoms (see the diagram on the right).
Because of these charges, polar molecules, like DNA or
RNA, can interact electrostatically with the water
molecules, allowing them to easily dissolve in water. Polar
molecules can therefore be described as hydrophilic and
non-polar molecules, which can’t easily interact with water
molecules, are hydrophobic. Nucleic acids are hydrophilic
due to the negatively charged phosphate (PO3-) groups
along the sugar phosphate backbone.
The role of the salt…
Ok, so back to the protocol. The role of the salt in the
protocol is to neutralize the charges on the sugar
phosphate backbone. A commonly used salt is sodium
acetate. In solution, sodium acetate breaks up into Na+
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
therefore much less soluble in water.
The role of the ethanol…
The electrostatic attraction between the Na+ ions in
solution and the PO3- ions are dictated by Coulomb’s Law,
which is affected by the dielectric constant of the solution.
Water has a high dielectric constant, which makes it fairly
difficult for the Na+ and PO3- to come together. Ethanol on
the other hand has a much lower dielectric constant,
making it much easier for Na+ to interact with the PO3-,
shield it’s charge and make the nucleic acid less
hydrophilic, causing it to drop out of solution.
The role of temperature…
Incubation of the nucleic acid/salt/ethanol mixture at low
temperatures (e.g. -20 or -80C) is commonly cited in
protocols as necessary in protocols. However, according to
Maniatis et al (Molecular Cloning, A Laboratory Manual 2nd
Edition… 2nd edition?? – I need to get a newer version!),
this is not required, as nucleic acids at concentrations as
low as 20ng/mL will precipitate at 0-4C so incubation for
15-30 minutes on ice is sufficient.
The wash step with 70% ethanol…
This step is to wash any residual salt away from the
pelleted DNA.
A few tips on nucleic acid precipitation…
• Choice of salt
o Use Sodium acetate (0.3M final conc, pH
5.2) for routine DNA precipitations
o Use Sodium chloride (0,2M final conc) for
DNA samples containing SDS since NaCl
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
precipitation and LiCl is more soluble in
ethanol than NaAc so will not precipitate,
but beware – chloride ions will inhibit
protein synthesis and DNA polymerase so
LiCl is no good for RNA preps for in vitro
translation or reverse transcription. In
these cases, use NaAc.
o Use Ammonium acetate (2M final conc)
for the removal of dNTPs, but do not use
for preparation of DNA for T4
polynucleotide kinase reactions as
ammonium ions inhibit the enzyme.
• To increase the yield in precipitations of low
concentration or small nucleic acid pieces
(less than 100 nucleotides)
o Add MgCl2 to a final concentration of
0.01M
o Increase the time of incubation ice before
centrifugation to 1 hour.
If you have anything to add, please feel free to leave a
comment!
http://bitesizebio.com/2007/12/04/the-basics-how-ethanol-
precipitation-of-dna-and-rna-works/
the alcohol does two things, changes the ionic potential of
the DNA and removes water molecules. this helps the DNA
to precipitate
Read more: Why use ethanol in DNA isolation | Answerbag
http://www.answerbag.com/q_view/1114236#ixzz10ctdxBx
7
Ethanol has a lower dielectric constant than water (it is less
electronegative by a fair margin), so when mixed with a
pure water/ DNA sample, it effectively lowers the dielectric
constant of the solution. When monovalent cations are also
added to the solution, the hydration shell that is keeping
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
(precipitates).
This is how ethanol (working with cations) works to
precipitate DNA.
Read more: Why use ethanol in DNA isolation | Answerbag
http://www.answerbag.com/q_view/1114236#ixzz10ctnh1z
Y
Ophiopogon japonicus (Mondo grass, Fountain plant,
monkey grass; Japanese: リュウノヒゲ ryu-no-
hige ("dragon's beard") or ジャノヒゲ ja-no-hige
("snake's beard")) is a species of Ophiopogon
native to Japan.
It is an evergreen, sod-forming perennial plant. The leaves
are linear, 20–40 cm long. The flowers are white to pale
lilac, borne in a short raceme on a 5–10 cm stem. The fruit
is a blue berry 5 mm diameter.[1]
[edit] Medicinal uses
In Chinese medicine Ophiopogon japonicus tuber, known as
mai men dong (Chinese: 麥門冬), is the cardinal herb for yin
deficiency. According to the Chinese Herbal Medicine
Materia Medica, the herb is sweet, slightly bitter and
slightly cold, enters the Heart, Lung and Stomach channels
and nourishes the yin of the Stomach, Spleen, Heart and
Lungs and clears heat and quiets irritability. It is used for
hacking dry coughs, dry tongue and mouth and
constipation. Liriope spicata is used as a substitute.[2]
[edit] Characteristics
It is also grown as an ornamental plant, providing an
excellent groundcover. Several cultivars have been
selected, including 'Albus' (white flowers), 'Compactus' and
'Kyoto Dwarf' (dwarf forms, not over 4–5 cm tall), and
'Silver Dragon' (variegated, with white-striped leaves). It is
often sold as a decorative plant for freshwater aquaria, but
because it is not a true aquatic plant, it may flourish for a
few months and then die. While hardy to temperatures of
about -20 °C when dormant in winter outdoors in normal
soil, when kept fully submerged it requires water
temperatures of 18-25 °C. It grows well in full sun or partial
shade. Propagation is from side shoots.[1][3]
The purpose of TE buffer is to protect DNA or RNA from
degradation. It is a buffer for storage of DNA & RNA.
To degrade DNA requires metal ions. THe EDTA in TE binds
the metal ions and so slows down the degradation. The
higher pH of the Tris also slws down DNA degradation.
TES buffer is a solution made up of Tris, EDTA and NaCl. Its
primary purpose to reduce the acidity of a solution. It is pH
stable and is also isotonic.
TE is Tris-EDTA pH 8.0.
Tris is needed to keep the pH at 8.0, which is the value at
which the double strand is stable (right pH for having H-
bonds) and EDTA chelates the 2+ ions, which are needed
for DNAses to cut DNA. In this way, DNAses cannot work.
Tris buffer maintains the pH.. EDTA prevents DNAse
enzyme by acting as a bivalent cation chelator. So the
extracted DNA can be preserved perfectly using TE buffer...
Tris EDTA buffer inhibits nucleases that will degrade the
DNA, by chelating cations required by the enzymes.
Phosphate buffer offers no such protection against
degradation.
uestion: What is a DNA pellet? How can one define this
term
logically and scientifically?
This refers to the mass of DNA at the bottom of the
centrifuge tube after
you do a DNA extraction. DNA is insoluble in ethanol, so
when you add
ethanol to a mixture containing DNA, the DNA will
precipitate in the
ethanol. Centrifuging will leave a 'pellet' of DNA.
Hope this helps,
Burr
I am assuming you are extracting DNA and it wants you to
pellet the DNA.
One molecule of DNA is too small to see with the even the
most powerful
microscope. In order to see it you need millions of strands
wrapped
together. If you then spin this in a centrifuge it will form a
visible mass
in the bottom of the tube. It will appear as a white dot in
the bottom of
the tube, also known as a pellet.
Vanhoeck
A DNA pellet is a clump of DNA that has been precipitated
from an aquaeous
solution by the addition of alcohol.
Ron Baker, Ph.D.
Do you need RNA? An RNAse treatment should get rid of
some of the sticky-ness. Also try incubating it overnight at
4 C (though if you've been working on it over the weekend,
you've probably done this anyway). One of the (many)
gDNA protocols I have for plant DNA extraction has the
final resuspension in R40 buffer (40ug/ml RNAse A in 1 x
TE), which you leave it in overnight at 4 C. Sometimes I like
to incubate it on a gentle rocking platform too, I don't know
if that helps, but it gives me the illusion that I'm doing
something while it's just hanging around in the cold room
Whatever you do, do not vortex it! It will shear the DNA.
I also find that freezing it at -20C then thawing it on ice can
sometimes help with the resuspension, though you don't
want to do it more than necessary (see above re:
shearing).
How long is your 70% EtOH wash? Sometimes increasing
that will help too.
And don't underestimate the amount of DNA you have. The
spec reading wont be accurate until the DNA is in solution -
better to resuspend it in a greater volume, find out that it's
too dilute then re-precipitate it than to mess about with it
at an unknown concentration.
Too much DNA for the TE volume or too dry as said before.
If too much DNA add a little more of TE and heat the
samples in a water bath at 55C for an 1h.
Are you sure that it's DNA?
Aside from using enough solvent to dissolve the DNA, DNA
is extremely soluble in aqueous solution. I wouldn't expect
you to have any problem in dissolving DNA in an
appropriate volume of TE buffer.
I agree with Astillius. Don't assume the DNA is insoluble
because you still see the pellet. The pellet is probably
some other crap. But if you are measuring solubility by
absorbance, then you may have a problem (no DNA in
pellet or insoluble). You might try placing it in a sonicating
water bath.
If you’re performing DNA/RNA precipitations, you will have
read Suzanne’s excellent article on which alcohol to use for
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
from a post-doc many years ago, one of those ‘magic-
hands’ scientists where everything seemed to work first
time. This tip of his is on the removal of the 70% ethanol in
the final step of the precipitation process.
The usual wisdom is to carefully drain off the ethanol and
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-
vac can be too fast meaning that you risk over-drying the
pellet making solubilisation more difficult.
So here’s the tip: After draining or pipetting off the 70%
ethanol, simply:
• recap the tube
• pulse the centrifuge to bring down the remaining
ethanol
• remove this liquid with a pipette and 200ul tip –
you can get right alongside the pellet if visible.
Leave the tube open as you move to the next
sample.
By the time you’ve finished your series of tubes (even if n=
3), the pellets are dry always enough to add diluent
immediately. Job done. This saves time and give you
samples are just the right degree of “dry”, every time.
Any other tips for ethanol (or even isopropanol!)
precipitation would be great to hear. Meanwhile – thanks
Michael.
DNA Precipitation
[edit] Theory
DNA is polar due to its highly charged phosphate
backbone. This polarity, based on the principle of "like
dissolves like", makes it soluble in water, which is also
highly polar. The high polarity of water, reflected by high
value of its dielectric constant 80.1 (at 20 °C), means that
electrical force between any two charges in aqueous
solutions is highly diminished compared to force in vacuum
or air.
This relation is reflected in Coulomb's law, which can be
used to calculate force acting on two charges q1 and q2
separated by a distance r, the dielectric constant (also
called relative static permittivity) of the medium is present
in the denominator of the equation ( is an electric
constant):
At an atomic level this diminishing of force acting on
charges results from water molecules forming hydration
shells around them. It makes water a very good solvent for
charged compounds like salts. Electric force which
normally holds salt crystals together by way of ionic bonds
is weakened in the presence of water allowing ions to
separate from the crystal and spread through solution.
The same mechanism operates in the case of negatively
charged phosphate groups on DNA backbone, even if
positive ions are present in solution, the relatively weak
electric force prevents them from forming stable ionic
bonds with phosphates and precipitating out of solution.
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
enough ethanol is added electrical attraction between
phosphate groups and any positive ions present in solution
becomes strong enough to form stable ionic bonds and
precipitate DNA. This usually happens when ethanol makes
around 64% of the solution. As the mechanism suggests
solution has to contain positive ions for precipitation to
occur, usually Na+, NH4+ or Li+ play this role [1].
[edit] Practice
DNA is precipitated by first ensuring that the correct
concentration of positive ions is present in solution (too
much will result in a lot of salt co-precipitating with DNA,
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
point but this has been shown to lower precipitation
efficiency [2][3]. The best efficiency is achieved at room
temperature but when possible degradation is taken into
account it is probably best to incubate DNA on wet ice.
Optimal incubation time depends on the length and
concentration of DNA. Smaller fragments and lower
concentrations will require longer times to achieve the
same recovery. For very small lengths and low
concentrations over-night incubation is recommended. In
such cases use of carriers like tRNA, glycogen or linear
polyacrylamide can greatly improve recovery.
During incubation DNA and some salts will precipitate from
solution, in the next step this precipitate is collected by
centrifugation in a microcentrifuge tube at high speeds
(~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
centrifugation. Centrifugation can be done either at room
temperature or in 4 °C or 0 °C. During centrifugation
precipitated DNA has to move through ethanol solution to
the bottom of the tube, lower temperatures increase
viscosity of the solution and larger volumes make the
distance longer, so both those factors lower efficiency of
this process requiring longer centrifugation for the same
effect.[2][3] After centrifugation the supernatant solution is
removed, leaving a pellet of crude DNA. Whether the pellet
is visible depends on the amount of DNA and on its purity
(dirtier pellets are easier to see) or the use of co-
precipitants.
In the next step, 70% ethanol is added to the pellet, and it
is gently mixed to break the pellet loose and wash it. This
removes some of the salts present in the leftover
supernatant and bound to DNA pellet making the final DNA
cleaner. This suspension is centrifuged again to once again
pellet DNA and the supernatant solution is removed. This
step is repeated once.
Finally, the pellet is air-dried and the DNA is resuspended
in water or other desired buffer. It is important not to over-
dry the pellet as it may lead to denaturation of DNA and
make it harder to resuspend.
Isopropanol can also be used instead of ethanol; the
precipitation efficiency of the isopropanol is higher making
one volume enough for precipitation. However, isopropanol
is less volatile than ethanol and needs more time to air-dry
in the final step. The pellet might also adhere less tightly to
the tube when using isopropanol[1].
[edit] Protocol
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
sodium acetate) of at least 95% ethanol.
3. Incubate on ice for 15 minutes. In case of small
DNA fragments or high dilutions overnight
incubation gives best results, incubation below 0
°C does not significantly improve efficiency [2][3].
4. Centrifuge at > 14,000 x g for 30 minutes at room
temperature or 4 °C.
5. Discard supernatant being careful not to throw out
DNA pellet which may or may not be visible.
6. Rinse with 70% Ethanol
7. Centrifuge again for 15 minutes.
8. Discard supernatant and dissolve pellet in desired
buffer. Make sure the buffer comes into contact
with the whole surface of the tube since a
significant portion of DNA may be deposited on the
walls instead of in the pellet.[1]
DNA is insoluble in alcohol, so using ethanol causes the
DNA to precipitate out. The colder the ethanol, the less
soluble the DNA is.The colder the ethanol is the greater the
amount of DNA that is precipitated.
Since our most popular article of all time (“The Basics: How
Ethanol Precipitation of DNA and RNA Works”) was
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
today, I’ll meet the challenge and discuss the pros and
cons of ethanol vs. isopropanol.
First, let’s review what we know about what is needed for
precipitation of DNA or RNA with ethanol:
1. Salt to neutralize the charge on the nucleic acid
backbone, causing the DNA to become less hydrophilic and
fall out of solution.
2. Ice to chill the sample. Lower temperatures promote the
flocculation of the nucleic acids so they form a larger
complex that readily pellets under the centrifugal forces of
a microcentrifuge.
3. A nucleic acid concentration high enough to force the
DNA out of solution (if the conc is not high enough, you can
add a carrier nucleic acid or glycogen to enhance the
recovery).
4. Centrifugation to pellet the sample
The difference between isopropanol and ethanol is
the solubility of DNA in each solvent.
DNA is less soluble in isopropanol so it will fall out of
solution faster and at a lower concentration, but the
downside is that the salt will too. With ethanol, the DNA
needs to be at a higher concentration to flocculate but the
salt tends to stay soluble, even at cold temperatures.
DNA falls out of solution in 35% isopropanol and 0.5M salt.
Using ethanol, the final concentration needs to be around
75% with 0.5M salt. So for the typical precipitation
protocol, isopropanol is added from between 0.7-1 volumes
of sample and ethanol is added at 2-2.5 volumes of
sample.
The choice of which solvent to use depends largely
on the volume of sample you need to precipitate.
If you are precipitating small volumes of DNA, and you can
fit the required amount of solvent into the sample tube,
then ice cold ethanol is the preferred choice. You can chill
it (some people use liquid nitrogen or -80C to accelerate
the precipitation) and precipitate more DNA without the
salt contamination that would occur from chilling
isopropanol. Afterwards you need to wash the pellet with
70% ethanol to remove salt.
Isopropanol use useful for precipitations where you have a
large sample volume (e.g. the eluate you get after using a
Qiagen plasmid Maxi Kit) because less solvent is needed,
so you can fit the whole lot in the (15ml) tube. But because
salts are generally less soluble in isopropanol than in
ethanol, they have more of a tendancy to co-precipitate
with the DNA. So to lessen the chances of salt
precipitation, isopropanol precipitations are carried our at
room temperature with minimal incubation times. Once
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
volatile ethanol. It is ok to chill the isopropanol precipitated
sample, if you are sure that it is not excessively salty.
Because DNA is less soluble in isopropanol, isopropanol
allows precipitation of larger species and lower
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
pelleting, to reduce the amount of salt you carry over.
So how do you choose when to use isopropanol and
when to use ethanol?
Use ethanol if:
You have room to fit two volumes of ethanol to sample in
your tube
1. The sample needs to be stored for a long period of
time and will be chilled
2. You need to precipitate very small pieces of DNA or
you have a very low concentration of sample so
you want to chill it longer and colder.
Use Isopropanol if:
1. You have limited in space in your tube and can fit
only 1 volume of sample
2. You need large molecular weight species because
incubation at room temperature for short periods
of time will not be conducive to precipitating small
species of nucleic acid
3. You are in a hurry and want to accelerate the
precipitation of nucleic acids at room temperature
What do I prefer? I use ethanol over isopropanol for most
cases, but will use isopropanol if I need to make everything
fit in one tube. My preferred protocol is 2 volumes of
ethanol and freeze at -20C for at least an hour or overnight
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
and keep my eye on the pellet when I decant everything.
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
when decanting the ethanol!
If I use isopropanol, I avoid cold temperatures because of
the excess salt that usually comes down with it. If I want to
increase the yields precipitated, I prefer to leave it
incubating at room temperature longer vs. chilling the
sample. When the DNA is pelleted, the pellet is sometimes
more difficult to see compared to the ethanol pellet. It can
be clear and glassy. Make sure, again, to note the side of
the tube where the pellet should be. Look for it before
decanting the isopropanol and 70% ethanol wash. After
washing with ethanol, the pellet becomes visible and white.
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
speed vac dry (5 minutes is enough) and then resuspend in
buffer.
Finally, for dry DNA pellets, heating the sample in buffer at
50-60C will help the DNA dissolve faster and won’t damage
the DNA. For RNA, heating can be used too (in water) at
temps around 42C. Overdried DNA and RNA will take
longer to dissolve so make sure not to speed vac for too
long.
Genes, or “coding DNA,” are segments of DNA that contain
the chemical recipe that determines particular traits.
Scientists now estimate that rice has about 37,500 genes,
located along threadlike, tightly coiled strands of DNA
called chromosomes. Genes, however, are only about three
percent of rice DNA; the rest is "noncoding" DNA. These
noncoding regions of the genome contain the information
that determines when and where genes are active – for
example, in which cell types and at what stages in growth
and development of rice.
More than $15 million was competitively awarded by NIFA,
NSF, and DOE, with more than $6.5 million of NIFA funding
awarded to The Institute for Genomic Research (TIGR) and
the University of Arizona. Researchers at the University of
Arizona collaborated with Cold Spring Harbor Laboratory in
New York, Washington University, and the University of
Wisconsin. These groups coordinated their research with
privately funded U.S. groups, including Rutgers University,
Monsanto, and Syngenta.
The finished genome sequence has already led to the
discovery of specific gene functions, which may help in
fighting diseases and physiological stress. It may also lead
to improved rice breeding, including the production of
higher quality rice in less time.
Rice has the smallest genome of all cereal grass crops and
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
wheat, barley, sorghum, and corn.
Rice Genome Fully Mapped
First DNA Map for a Crop Expected to Boost
Modification Efforts
By Justin Gillis
Washington Post Staff Writer
Thursday, August 11, 2005
Scientists have completed a genetic map of the rice plant,
a scientific milestone that they hope will accelerate efforts
to feed the hungry by improving the world's most
important food.
Rice is the first crop plant whose complete genetic
sequence, or genome, has been compiled and placed in
computer data banks around the world. It will be a key tool
for researchers working on improved strains of rice and
other grains as they struggle to stay ahead of human
population growth. A paper describing the genome is being
published today in the journal Nature, and the sequence
will be freely available to researchers worldwide.
Centrifuge to separate the DNA from the dissolved salts
and sugars
DNA EXTRACTION
Protocol and notes
Introduction
• This highlights a simple protocol for extracting DNA
from cereal plant leaves
• This protocol will produce a product that is suitable
as a template for polymerase chain reaction (PCR) and
restriction enzyme digestion
• It will also contain RNA which can be removed with
further purifications
DNA Extraction
Protocol Overview
• Fresh plant material is collected
• The plant material is mixed with extraction buffer
and ground in a mortar and pestle
• The resulting liquid is centrifuged to separate the
solid plant material from the extraction buffer supernatant
• The supernatant is mixed with ethanol to
precipitate the DNA which will form a pellet at the bottom
of the tube
• The supernatant is discarded
• The DNA pellet is washed with a diluted ethanol
solution
• The DNA pellet is dried and then dissolved in water
or a buffer
Protocol for Collecting the Plant Material
• Set up and label (according to species) TWO
eppendorf tubes for each seedling you will be extracting
DNA from.
• Add 600 µL of isopropanol to one tube in step 1
and leave the other tube empty.
• Add 2.0 mL of extraction
buffer to the mortar.
• Cut a 2 inch portion of leaf
from each plant and grind
in the mortar with the pestle
until the solution is deep green.
Transfer 1.5 mL of solution to empty tube.
• 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