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The Bitesize Book of

Know-How for Nucleic Acid
Extraction and Purification
Table of
Contents

1. An Introduction to Nucleic
3
Acid Extraction and Purification

1.1 Why Isolate Nucleic Acids? 4

1.2 Cell Lysis Methods 5

1.3 Underlying Principles of

8
Nucleic Acid Purification

1.4 Critical Factors and How

14
They Influence Success

1.5 Special Situations 15

2. Assessing Your Isolated

16
Nucleic Acids

2.1 Quantification and Quality

16
Control

2.2 Improving Nucleic Acid

20
Quality
 Nucleic Acid Estraction and Purification
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1. An Introduction to Nucleic
Acid Extraction and Purification
Nucleic acid extraction is a fundamental part of
molecular biology because high quality nucleic
acids are critical to most applications. In this guide,
we will provide an overview of the currently used
techniques for nucleic acid isolation and some tips to
help you choose the right technique for your sample
type. We will cover the ways in which you can assess
purified nucleic acids for quantity and quality, and
we will introduce the most commonly encountered
problems during nucleic acid extraction.
New England Biolabs is a
recognized world leader in
the discovery, development,
and commercialization of
recombinant and native enzymes
for genomic research.
 Nucleic Acid Estraction and Purification ww.neb.com

1.1 Why Isolate Nucleic Acids?

Isolated nucleic acids provide answers to

a plethora of research questions across a
broad range of applications (e.g., cloning,
qRT-PCR, and genome- or transcriptome-
wide next generation sequencing). The
information obtained from isolated
nucleic acids can be used in a number of
ways.

The exact research goal determines the

type of nucleic acid to be extracted and
the application often influences the choice
of extraction method. For example, a
standard end-point PCR reaction does
not require the same DNA quality as a
whole-genome sequencing experiment.
Reasons for isolating nucleic
acids
1. To analyze gene expression for basic or
disease-oriented research.

2. To follow responses to medical

To identify the best extraction method
for your work, it is necessary to have a
clear understanding of the downstream
application, as well as any potential
limitations associated with your sample
type (e.g., clinical samples are often
limited in amount and challenging to
work with). Depending on the sample
type, the cell lysis method may differ,
but the overall nucleic acid extraction
concept will remain the same: cells or
tissue samples are lysed and non-nucleic
contaminants (e.g., proteins) are removed,
before the nucleic acids are washed and
concentrated.
treatments (e.g., monitoring of viral
titres during and after anti-viral
therapy).
3. To identify new species and to gain a
deeper understanding of evolutionary
processes (e.g., ancient DNA analysis).
4. To monitor and type pathogens
responsible for infectious disease
outbreaks in humans, animals, and
plants.
5. To monitor food and water safety
via microorganism detection and
quantification.

6. To diagnose diseases (e.g., genetic

disorders, cancer, immunological
deficiencies).

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1.2 Cell Lysis Methods Info

An over v iew of t he most
The cell lysis method used depends to a large extent commonly used lysis met hods
for a ra nge of sa mple t y pes is
on the sample type, with tougher tissues like plants presented in Table 1.
requiring more force than mammalian cells.

Cell lysis methods are grouped into 3 main categories:

mechanical, enzymatic, and chemical lysis. The
principles underlying the 3 lysis approaches and the
pros and cons of each are presented below:

1.2.1 Mechanical Lysis

In mechanical lysis, cellular membranes are disrupted
by an external force (see Table 1 for examples).

Pros
• Often efficient and fast
• Fast lysis reduces time between harvesting the
sample and the isolation of nucleic acids, which
might be crucial in gene expression analysis
experiments
• Ideal for tough-to-lyse samples (e.g., plant material,
filamentous fungi, and yeast)

Cons
• Time-consuming when processing multiple
samples, depending on method used (e.g., pestle
and mortal processing is very slow)
• Slow processing of large sample numbers may
increase the risk of sample degradation
• Can generate heat in a sample, leading to protein
aggregation and nucleic acid degradation
• Requires special equipment/tools

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 Nucleic Acid Estraction and Purification
1.2.2 Enzymatic Lysis
In enzymatic lysis, an enzyme is added to digest
proteins or cellular structures, such as cell walls
of yeast and filamentous fungi.
Pros
• Ideal in a lab that doesn’t possess mechanical
lysis equipment
• Selective removal of the cell wall leaves
behind the remainder of the cell for
another lysis method (usually chemical).
This provides flexibility and circumvents
some of the damage that may be caused by
mechanical lysis
Cons
• Can be time-consuming (enzymatic
digestion may take 1 hour) and expensive
• Not ideal for gene expression analysis as
enzyme-induced cellular changes may
impact gene expression
Next Page
Table 1: Over v iew of Commonly
Used Lysis Met hods. Note: It is not
possible to list ever y sa mple t y pe
here, so if your sa mple t y pe doesn’t
appear in t he table, consu lt your
col leag ues or suppliers to f ind t he
correct lysis met hod.
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1.2.3 Chemical Lysis
During chemical lysis, cells are washed with a
detergent that breaks down lipid membranes,
thus releasing cellular components. In addition
to a detergent, chemical lysis buffers usually
contain chaotropic salts, such as guanidine
hydrochloric acid or urea, which help destabilize
proteins (e.g., nucleases) that might degrade
the newly exposed nucleic acids, and prime the
nucleic acid for binding to silica-matrixes. The
exact composition of a chemical lysis buffer
varies depending on the application and sample
type.
Pros
• Cheap, easy, fast to perform
• No specialized equipment required
Cons
• Detergents that disrupt the cell membrane
will often lyse other intercellular membranes
as well, and thereby releasing their
components. This approach isn’t readily
suitable for organelle-specific nucleic acid
extraction
• While this technique works well for E. coli,
it is not effective for gram-positive bacteria,
plant cells, or fungal cells because of the
presence of hard cell walls that prevent the
detergent from accessing the cell membrane
• Adding both a detergent and chaotropic salt
to an E. coli sample may compromise the
ability to differentiate plasmid DNA from
genomic DNA. However, steps can be taken
to differentiate these, which will be discussed
later
• Harsh chemicals can present a danger to the
researcher
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Category 1: Mechanical Lysis

Method Description Sample Ty pe

Blender blades grind sa mples Yeast, complex tissue sa mples

Waring Blender
at high speed (e.g., liver, muscle)

Grinds sa mples whi le leav ing

Dounce Homogenizer Cu ltured cel ls
orga nel les intact

Meta l beads are agitated w it h Ma mma lia n, pla nt,

Bead Beater
sa mples at high speed microorga nisms

Sa mples are ground w it h a

Ma nua l grinding w it h mor tar
mor tar a nd pest le in liquid Pla nt, f i la mentous f ungi
a nd pest le
nitrogen

Sa mples are subjected to

Freeze-Thaw Bacteria
mu ltiple f reeze/t haw c ycles

Sa mples are disrupted as t hey

French Press are forced t hrough a sma l l Pla nt, f i la mentous f ungi
space

Sa mples are broken dow n

Sonication w it h high f requenc y sound A l l sa mples t y pes
waves

Category 2: Enzymatic Lysis

Method Description Sample Ty pe

Enz y mes t hat target gluca n

Lysostaphin a nd chitin, respectively, in Staph lococcus spp.
f unga l cel l wa l ls

Targets gluca n a nd chitin,

Gluca nase, chitinase respectively, in f unga l cel l Yeast, f i la mentous f ungi
wa l ls

Enz y me t hat brea k s dow n

Ma mma lia n, pla nt,
Cel lu lase cel lu lose, a major component
microorga nisms
of pla nt cel l wa l ls

Enz y me used in conjunction

Maceroz y me w it h cel lu lase to brea k dow n Pla nt
pla nt cel l wa l ls

Sa mples are subjected to

Freeze-Thaw Pla nt
mu ltiple f reeze/t haw c ycles

Category 3: Chemical Lysis

Method Description Sample Ty pe

Series of chemica l washes to

A l ka line Lysis ex tract nucleic acids f rom Bacteria l plasmids
bacteria l cel ls

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 Nucleic Acid Estraction and Purification
1.3 Underlying
Principles of Nucleic
Acid Purification
After cell lysis, nucleic acids
are selectively isolated from the
surround cellular components,
concentrate, and stored in water
or a suitable buffer for use
in downstream applications.
The steps following cell lysis
are collectively referred to as
purification. Since the chemistry
of the isolated nucleic acid
remains the same regardless of
sample type, the purification
strategies described below are
generally applicable to all sample
types.
1.3.1 Spin Columns
Often referred to as “using the
kit”, spin column extraction
and purification allows rapid
purification and cleanup of high
quality genomic DNA, plasmids,
RNA, and PCR products, without
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the need to make fresh solutions.
Spin columns contain a solid
matrix of silica or proprietary
resin for selective binding of the
nucleic acid of interest.
A Typical Spin Column
Protocol:
1. Application of lysed sample to
spin column
The lysed sample, collected in
a binding buffer containing
chaotropic salts, is applied to
a spin column. The binding
buffer allows the nucleic acids to
disassociate from the aqueous
solution and bind to the column
matrix.
2. Nucleic acid binding
Centrifugation brings the entire
sample into contact with the
column matrix. Nucleic acids in
the sample selectively bind to
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the column, while other cellular
components pass through (this is
often called the flow-through).
3. Washing
After binding, the column is
washed to remove any remaining
contaminants like protein or
residual salts that may severely
impair downstream applications.
The number of washes performed
is kit-dependent. Some wash
buffers contain chaotropic salts
to remove protein, and some have
a high ethanol concentration for
salt removal. Most kits include a
final wash step in a buffer almost
entirely comprised of ethanol to
ensure complete salt removal.
4. Spin to removal residual
ethanol
After washing, a short dry spin is
sometimes performed to remove
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residual ethanol. to rehydrate the nucleic acids, nuclease activ it y. W hi le TE

5. Elution
To elute the nucleic acids from
the matrix, a small volume of
water or elution buffer* is added
and the column is allowed to
rest for a few minutes. Since
chaotropic salts have been
removed in Step 4, the aqueous
elution buffer will now be able
disassociating them from the
column matrix. A final spin
allows this aqueous solution
containing the nucleic acid
sample to be transferred to a
fresh tube.
* Nucleic acids are usua l ly
stored in a buf fer conta ining
Tris a nd EDTA (TE buf fer). The
presence of EDTA helps to inhibit
buf fer is ef fective in inhibiting
nucleases, t he a mount of EDTA
present in TE is genera l ly orders
of magnitude lower t ha n t he
a mount of Mg 2+ present in most
enz y matic reactions, so t he
presence of EDTA in t he elution
buf fer shou ld not be a concern.

Pros and Cons of spin column kits Pros Cons

• Cost effective • Slow growing

• High efficiency strains can pro-
• Reliable duce lower yield
• Yields nucleic • Nucleic acid loss
acids suitable for caused by incom-
use in downstream plete elution
applications • Yield is limited by
binding capacity
of column

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 Nucleic Acid Estraction and Purification
1.3.1.1 A Note on Plasmid Spin Kits
Plasmid miniprep kits are among the most
widely used kits in molecular biology labs,
allowing for rapid plasmid isolation from E.
coli. Since E. coli is amenable to chemical lysis,
plasmid kits combine sample lysis and nucleic
acid purification as follows:
1. Bacterial cells are pelleted and lysed in
microcentrifuge tubes, before the cellular debris
is pelleted by centrifugation.
2. The lysis solution is formulated so that only
the plasmid DNA binds to the spin column
after lysis**. Therefore, the cell lysate can be
loaded onto a column immediately following
centrifugation.
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3. After binding of the plasmid-containing lysate
to the spin column, the remaining purification
steps are similar to all other spin column
protocols.
4. Ready-to-use high quality plasmid DNA is
eluted in water or TE buffer.
**Most spin kits differentiate plasmid DNA
from genomic DNA (gDNA) during the lysis
step. The lysis buffer contains sodium hydroxide
and sodium dodecyl sulfate, which completely
denature plasmid and gDNA. The proceeding
step neutralizes the sample, allowing the plasmid
DNA to reanneal. High molecular weight gDNA,
however, cannot fully reanneal and tends to
tangle with proteins present in the sample,
preventing the gDNA from binding the spin
column, thus leading to its removal. Despite their
efficiency and reliability, plasmid spin kits come
with a few drawbacks. Since they are optimized
for use with standard E. coli strains, slow growing
or unstable strains will likely produce low yields
if used with standard protocols. Furthermore,
plasmid miniprep kits, and spin kits in general,
are limited in binding capacity, with a total
yield of between 50 to 100 μg, depending on
the kit. Also, the DNA isolated with these kits
generally has higher endotoxin levels. For routine
transfections where larger yields and endotoxon-
free DNA are required, it is worth considering a
midi- or maxiprep kit, which use spin columns
with larger loading and binding capacities.
 Nucleic Acid Estraction and Purification ww.neb.com

1.3.1.2. Other Spin Column Kits

In addition to nucleic acid extraction,

spin columns are also used for nucleic
acid cleanup and concentration. Cleanup
kits can be used to remove unused
PCR reagents following PCR and other
enzymatic reactions, and to purify DNA
from agarose gels. Concentrating your
sample can provide more flexibility with
many downstream applications.

Some commercial spin column kits have

size-selection capabilities, allowing you to
select the fragment size range you want
applications. Finally, Monarch kits allow
to study (e.g., small RNA species). This is
small volume elution, resulting in more
accomplished by altering the amount of
concentrated nucleic acids and eliminating
alcohol present in the buffer system.
the need for downstream concentration
steps.
Many modern spin kits combine cell
lysis and nucleic acid purification, and
nowadays you can find a spin column for
nucleic acid isolation from almost any
sample type, including, but not limited
to, insects, plants, seeds, fungi, bacteria,
saliva, blood, feces, and FFPE-samples.

NEB has recently released the Monarch

line of nucleic acid purification kits. This
line of spin column kits is specifically
designed to reduce environmental impact
by including thin-walled columns, which
contain reduced amounts of plastic.
Additionally, these kits ensure less buffer
retention, resulting in higher purity nucleic
acids, for greater results in downstream

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 Nucleic Acid Estraction and Purification
1.3.2 Phenol/Chloroform and Ethanol
Precipitation
Phenol/chloroform extraction is a manual
method that relies on the principle of differential
solubility to isolate nucleic acids. Samples are
exposed to a mixture of phenol and chloroform
in a given ratio, depending on the desired
nucleic acid. Protein is soluble in phenol/
chloroform, and nucleic acids are water-soluble.
When the phenol/chloroform solution is mixed
with the sample, proteins and nucleic acids are
separated, paving the way for purification.
For dual extraction of RNA and DNA, this
procedure can be adjusted by the addition of
acid phenol. This renders DNA uncharged as a
result of the excess H+ ions interacting with the
phosphate backbone. DNA will then be soluble
in the phenol layer of the phenol/chloroform
extraction, while RNA will remain soluble in
the aqueous phase, owing to its naturally more
acidic nature. The aqueous and organic phases
can be separated after centrifugation and each
can be subjected to ethanol precipitation, as
described above.
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A Typical Phenol/Chloroform Extraction
Protocol:
1. Exposure to phenol and chloroform:
Previously lysed samples are mixed with
phenol/chloroform, usually by strong
vortexing. Vortexing ensures that all organic
components can fully interact with the
phenol/chloroform mixture for complete
solubilization and removal.
2. Centrifugation: After Step 1, two phases are
visible. The aqueous phase containing the
nucleic acids sits at the top, while the organic
phase on the bottom contains proteins,
lipids, and other macromolecules. The
sample is then centrifuged to fully separate
the two phases.
3. Phase separation: The aqueous phase is
carefully removed by pipetting.
4. Ethanol precipitation: The nucleic acids are
purified via ethanol precipitation. During
ethanol precipitation, salts and ethanol are
added to buffer the nucleic acids from the
water solution. The salts buffer the sugar
phosphate backbone, and the ethanol alters
the solution’s dielectric constant. This
allows the nucleic acids to separate from the
aqueous solution, permitting subsequent
isolation by high-speed centrifugation.
5. Resuspension: Pelleted DNA or RNA is
resuspended in water or TE buffer.
 Nucleic Acid Estraction and Purification
Pros and Cons of phenol/chloroform extraction:
Pros
• High efficiency, often resulting in higher
yield than spin kits
• Suitable for extraction of intact high
molecular weight DNA (e.g., gDNA)
• Samples that are suspended in complex
solutions are usually still amenable to
the procedure, whereas some volatile
compounds can interfere with a spin column
matrix
• For fatty samples (e.g., brain tissue), phenol/
chloroform extraction is superior over most
spin column kits
1.3.3 Automated Methods for Higher
Throughput
Depending on the application and sample
type, there may be high-throughput techniques
available for your application. One of the most
commonly used high-throughput techniques
is the use of magnetic bead separation. In this
technique, positively charged magnetic beads
are introduced to the sample. DNA will bind to
the positively charged beads at low pH and will
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Cons
• Phenol and chloroform are harmful if
handled incorrectly, and must be treated
with extreme caution in fume hoods, as
well as be disposed of in appropriate waste
streams
• This method is much more time-consuming
than a spin kit, and may result in a lower
yield
• Traces of phenol and chloroform in resulting
nucleic acid extracts can negatively impact
downstream enzymatic reactions, like PCR.
If this is the case, the nucleic acids will need
to be cleaned up prior to PCR. A number of
spin column PCR cleanup kits exist for this
purpose
• It can take a bit of practice to be able to
confidently extract the aqueous phase
without contaminants
be released at high pH. Beads can be removed
through the use of magnets, and the pH of the
solution can be easily adjusted to isolate the
desired nucleic acid. The technique is fast and
efficient, but the investment in automation
equipment can be quite costly.
 Nucleic Acid Estraction and Purification
1.4 Critical Factors and How They Influence Success
When performing nucleic acid
extraction or purification, it is
important to pay close attention
to a number of factors such as pH,
salt concentration, temperature,
buffer volume, and potential ethanol
contamination. Each of these factors
can greatly impact yield, quality, and
success of downstream applications.
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• Suboptimal pH or salt
concentration can alter the
charge of the nucleic acid in
question, which could prevent
binding to a spin column, or lead
to solubility in the wrong phase
of a phenol/chloroform reaction.
• Incorrect buffer volumes
can lead to incomplete lysis,
neutralization, or undesired
dilution of the eluted nucleic
acids
• Ethanol contamination can
inhibit downstream enzymatic
reactions and cause your sample
to float out of an agarose gel well.
 Nucleic Acid Estraction and Purification
1.5 Special Situations
1.5.1 High Molecular Weight DNA
Extraction
For some applications (e.g., southern blotting and
some sequencing procedures), extraction of intact
high molecular weight (HMW) DNA is necessary.
To extract HMW DNA, additional factors need
to be considered in addition to the ones described
above:
• HMW DNA can easily fracture during
extraction. Exposure to shearing forces
(vortexing, sonication, etc.) can lead to the
breakdown of DNA molecules, resulting in
shorter fragments after extraction. To avoid
this, use an extraction and lysis protocol that
places minimal force on the sample.
• For applications in which visualization of
HMW DNA is necessary, it is possible to
perform lysis and extraction within agarose gel
plugs, thus stabilizing the DNA throughout the
procedure (e.g., pulsed-field gel electrophoresis),
where entire chromosomes are kept intact and
visualized by electrophoresis.
• Alkaline lysis often results in the loss of HMW
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DNA because it becomes irreversibly tangled
during the denaturation step.
• If using a spin column for HMW DNA
extraction, consider the binding size capacity
of the column matrix. Some columns are
only able to reproducibly purify 10 – 15 kb
sized fragments because larger fragments
are difficult to elute from the column due to
tight binding. For larger fragments, it may be
necessary to consider specialized columns for
high HMW binding, or a manual method such
as phenol/chloroform extraction and ethanol
precipitation.
1.5.2 Small RNAs
Small RNA molecules are often lost during
traditional RNA extraction procedures because
traditional spin columns usually have a lower size
limit of approximately 100 base pairs, though size
cut-offs are very dependent on buffer formulation.
Because small RNA molecules are extremely
important for understanding biological function,
most modern clean up kits for total RNA extraction
are optimized to capture these small RNAs.
 Nucleic Acid Estraction and Purification ww.neb.com

2. Assessing Your Isolated

Nucleic Acids

2.1 Quantification and Quality Control

Following nucleic acid extraction and

purification, it is important to understand the
quantity and quality of the extracted samples.
Depending on the downstream application,
fluctuations in these parameters can drastically
alter the outcome. For example, expression
analysis by qRT-PCR will result in unreliable data
if the exact same amounts of total RNA are not
used for each cDNA synthesis reaction.

There are a number of nucleic acid assessment

methods, which vary in time consumption, cost,
and precision. Ultimately, the method should be
chosen with the requirements of the downstream
application in mind. Here is an overview of some
of the most common assessment methods:

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 Nucleic Acid Estraction and Purification
2.1.1 Agarose Gel
Electrophoresis
Agarose gel electrophoresis
separates nucleic acids based
on their molecular weight
by pulling them through a
solidified gel matrix in the
presence of an electric current.
Extracted nucleic acids are
compared to a molecular
weight standard run in
parallel, containing fragments
of known sizes and quantity.
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Pros
• Visual determination of DNA quality
• Intact genomic DNA samples appear as bands that
barely migrate out of the well
• Degraded nucleic acid appears as a smear
• Ribosomal RNA can be clearly seen
• Available to most laboratories
• Allows you to visualize the presence of other potential
nucleic acid contaminants (e.g., the presence of RNA
in plasmid purification samples)
Cons
• Provides only a crude estimate of quantity
• Does not reveal the presence of contaminants, such as
salts, within a sample
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2.1.2 Capillary Electrophoresis

Capillary electrophoresis, sometimes referred

to as lab-on-a-chip, pulls samples through a
small capillary that is monitored by a detector. A
computer receives the information and displays
it graphically. Capillary electrophoresis is
suitable for analysis of fragment size, quantity,
and overall sample quality. This technology is
much more precise than traditional agarose
electrophoresis, and is often the method of
choice for nucleic acid assessment prior to qPCR.

Pros Cons

• Precise analysis of most types of nucleic • Expensive

acids

• Automated sizing and quantification is much

more exact than gel-based methods

• High sensitivity – can detect and analyze

even small amounts of sample

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2.1.3 Spectrophotometry

Spectrophotometry is a fast technique that relies on

the properties of light interacting with samples for
precise quantification. Samples are loaded into an
instrument (the spectrophotometer) that passes
light at varying wavelengths through them, which is
then picked up by a detector. The detector provides
information regarding sample quantity
and quality that is then translated via computer
software.

Absorbance measurements are taken at 260 nm and

280 nm, and the ratio of the two indicates the purity
of the sample. For pure DNA and RNA, the 260/280
ratio should be between 1.8 and 2.1. An additional
measurement can be taken at 230 nm, and a 230/280
nm ratio below 2.0 indicates the presence of organic
contaminants.

Recently, a new type of fluorescence-based

spectrophotometric analysis has become prevalent
in molecular biology labs. This fluorescence-
based technique achieves increased sensitivity
over traditional spectrophotometry, and is able to
distinguish between DNA and RNA via the use of
DNA- and RNA-specific fluorescent dyes.

Pros:
• Accurate and fast
• Provides information on the presence of
contaminants
• Spectrophotometer available to most laboratories

Cons:
• Inability to quantify individual fragments
• Expensive

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 Nucleic Acid Estraction and Purification
2.2 Improving Nucleic Acid Quality
Despite our best efforts, it is often necessary to take
additional steps to improve nucleic acid quality. The
following sections will describe some additional
factors to consider when improvements in nucleic
acid quality are needed.
2.2.1 Working and Storage Temperature
Nucleic acids are susceptible to degradation by
nucleases (i.e., RNases and DNases), and storage at
low temperatures helps inhibit this activity.
DNA is inherently more stable than RNA, and is
more resistant to nuclease activity at higher storage
temperatures. DNA should be stored at or below
-20°C, and kept on ice while in use. Bear in mind
that repeated freeze-thaw cycles may fragment
DNA, especially HMW species. HMW DNA
extracts should therefore be stored at 4°C if they are
used frequently.
RNA is much more susceptible to nuclease
degradation and should always be stored at very low
temperatures (-20 to -80°C), and only thawed when
absolutely necessary.
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2.2.2 Nuclease Inhibitors and Cleaning
Solutions
As mentioned previously, the presence of nucleases
can quickly degrade a nucleic acid sample. It is easy
to transfer nucleases from an ungloved hand to a
work surface; thus, gloves should always be worn
when working with nucleic acids.
Workspaces should be kept clean and frequently
wiped down with ethanol to remove nuclease
contamination. There are a number of
commercially available products to prevent RNase
contamination. Many researchers also add the
RNase-inhibitor diethyl pyrocarbonate (DEPC)
to water that is destined for RNA work. It is also
advisable to wash and treat glassware with DEPC
prior to RNA work.
In recent years, a number of nucleic acid stabilizing
reagents have appeared on the market. Unlike the
cleaning solutions described above, these reagents
are added directly to the intact sample at the point
of collection to inhibit nucleases and stabilize
nucleic acids until extraction is carried out. While
the majority of these reagents are compatible with
most downstream extraction kits and applications,
bear in mind that some of them require removal
from the intact sample prior to nucleic acid
extraction.
 Nucleic Acid Estraction and Purification ww.neb.com

encountered issues during nucleic acid

purification. The tips in NEB’s guide may
also be useful for kits from other commercial
suppliers, but it is advisable to consult the
supplier of your kit for the most precise
troubleshooting advice.

You may also find it useful to read

protocol-based scientific articles. The underlying
cause of suboptimal nucleic acid extraction is
often resolved easily when you fully understand
the underlying principles of the extraction
technique.

If you’ve followed the troubleshooting advice

from your kit’s supplier, and you can’t rectify the
issue, then it may be time to contact technical
2.2.3 Use Nuclease-Free Consumables support. Technical support representatives
are very knowledgeable and are likely to have
When working with any nucleic acid, ensure encountered other users of your kit with similar
that any consumables or glassware are treated issues.
for nucleases. Purchase nuclease-free tubes and
pipette tips with filters when possible.
If, following analysis, you find that your
DNA yield is poor, the quality is suboptimal,
or if the isolated DNA passes your quality
assessments but you achieve suboptimal results
in downstream applications, you will need to do
some troubleshooting.

Because there are many potential pitfalls,

there are also many troubleshooting steps.
NEB’s troubleshooting guide for nucleic acid
purification covers the many commonly

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