Ways and Tips for DNA

Extraction and Purification

CD

5 Ways to Extract and Purify A DNA Sample

Phenol-chloroform extraction

Phenol-chloroform extraction, also known as organic extraction, uses sodium dodecyl

sulfate (SDS) and proteinase K to enzymatically digest proteins and non-nucleic acid
cellular components. Then, a mixture of phenol: chloroform: isoamyl alcohol (25:24:1)
is added to promote the partitioning of lipids and cellular debris into the organic
phase while leaving isolated DNA in the aqueous phase. After centrifugation, the
purified DNA in the aqueous phase can be transferred to a clean tube for
downstream analysis. Alternatively, DNA can be recovered and concentrated from the
aqueous phase via ethanol precipitation or the application of a centrifugal filter unit,
so that the DNA in the samples can be further purified and concentrated.

Pros:
A cheap, reliable and effective way to
remove proteins from DNA solutions. DNA
Protein
Cons:
Cell Lipids
Time-consuming, usage of hazardous Cells Lysate DNA
chemicals, increased opportunities for
contamination and sample mishandling,
possibility of inhibiting downstream Add SDS and Add PCIA Transfer and Retain
enzymatic reactions due to Proteinase K Incubate Vortex and Centrifuge Aqueous Phase DNA
phenol/chloroform carry-over.

Tel: 1-631-624-4882 Email: info@cdparticles.org

Ethanol precipitation

Ethanol precipitation is a common way for desalting

Pros:
and concentrating DNA. Both monovalent cations
A cheap and effective way to desalt and
(0.1-0.5 M, usually in the form of the acetate salt of concentrate DNA.

sodium) and ethanol are added to the DNA to a final Cons:

Time consuming, risks of carrying
concentration of 70%. Ethanol could change the DNA ethanol over into the final sample.

structure, leading to the aggregation of the DNA

molecules and precipitation out of solution. Most salts and small organic molecules
are soluble in 70% ethanol and the precipitated DNA is left to be ready for separation
by centrifugation.

Silica column-based kits

Loaded
lysate

Pros:
Convenient, relatively fast, the ability to
Centrifuge
Centrifuge
process a large number of samples.
Silica
membrane Cons:
Relatively costly, low yields (as low as
25%), chaotropic salt carry-over.
Sample Column Wash and Dry the DNA
Lysis Loading Membrane Elution

It's a convenient approach to DNA cleanup. The principle is that chaotropic salts are
added to the sample to denature the DNA via disrupting its hydrogen bonds. Under
this condition, the DNA will selectively bind to the silica resin in the column and thus
promoting its separation from the rest of the sample. After washing, a low salt
solution is used to elute the DNA from the column, which makes the renaturing of the
DNA, leading to its decreasing affinity for the silica. Based on this technology, there
are many commercial kits which are ideal for DNA cleanup after applications such as
agarose gel extraction, enzymatic reactions and PCR.

Tel: 1-631-624-4882 Email: info@cdparticles.org

Anion exchange

The basic principle is that the positively charged DEAE Cell Extract

functionalized resins are able to bind the negatively

charged DNA phosphate backbone. By using specific
Salt More Salt
salt in specific pH condition, DNA in the sample will
bind the resin, followed by stringent washing steps to
remove contaminants such as protein and cellular
debris. After the above steps, DNA can be selectively Ion-exchange Resin

eluted from the resin.

Pros:
High purity DNA, ideal for downstream applications e.g. transfection and
DNA sequencing. Protein
and RNA
Cons:
Resins are expensive. Discard DNA

Magnetic beads

By using magnetic beads, DNA can conditionally bind

to them in a pH-dependent manner, which enables
you to isolate the DNA from the rest of the sample by
simply controlling pH. To be more specific, the
magnetic beads are positively charged and bind DNA Sample
Add
Magnetic
Remove the
Lysis Supernatant
Beads
at low pH, while at high pH they become negatively
charged and thus release the DNA. There are a few Direct Use

commercial magnetic bead kits available now.

DNA Elution

Pros:
Fast, no chaotrophic salts or organic washing solutions required, ideal for Wash

automation of high throughput processing.

Cons:
The initial cost in purchasing the magnets is reasonably high.
This procedure can be tricky when handling multiple samples without
an automated workflow.

Tel: 1-631-624-4882 Email: info@cdparticles.org

Tips for DNA Extraction and Purification with Magnetic Beads

As one of DNA cleanup options, magnetic beads are simple and effective to fulfill
molecular biologists' desire of DNA purification. However, even a simple technique can
go wrong and produce sub-optimal DNA end products. To help ensure that the simple
stays effective, we introduce you some tips for DNA extraction and purification with
magnetic beads.

Never freeze your magnetic beads

Unless otherwise mentioned, magnetic beads can never ever

be frozen. Beads must always be stored at 2 to 8 ℃. There may
be cracks on the surfaces of your beads after freezing and
thawing, leading to sample contamination.

Bring your beads to room temperature

Before using your magnetic beads, you should bring them to

room temperature. In other words, you need to plan ahead and 77°F 25℃
68°F 20℃
leave enough time (usually 30 minutes are ideal) for beads to
warm up before you need them.

Homogenize your magnetic beads

Magnetic beads are huge compared to the stuff in molecular biology. Therefore, they
may settle into the bottom during storage. However, you need a homogenous bead
slurry to conduct experiments, so you should vortex your beads immediately before
using them. You may not continue your experiments until the slurry is an even color
and all your beads are in suspension, or you may risk endangering your protocol.

Pay attention to DNA-bead ratio

Bead input is directly correlated to the amount of DNA that you

should obtain. Your sample might get contaminative with too
many beads, or you might lose product with too few beads. So,
you must pay attention to DNA-bead ratio based on the
protocol and do not modify randomly.

Tel: 1-631-624-4882 Email: info@cdparticles.org

Choose an appropriate magnetic stand

Whether you are using strip tubes, a 96-well plate, or a MIDI

plate, choosing appropriate magnetic stand matters. In
addition to the size, stands vary in their magnet placement.
Magnet placement affects the aggregation place of your
beads, such as the bottom of the well, to the left, to the right,
or in a ring conformation. If your protocol doesn't point out
which stand is best, you can choose based on your personal
preferences. Moreover, you'd better find a stand with good
magnetic strength.

Pipette carefully

Keep your fingers steady and go slow, don’t aspirate too quickly.

Monitor your loss

Keep an eye on your sample loss at each wash step, so that you may clarify
why some samples might work better than others.

Creative Diagnostics provides a variety of DNA extraction and purification products,

including silica coated magnetic beads , ion-exchange magnetic particles and
commercial purification kits. Please visit our website to explore more.

For more information,

view our website: www.cd-bioparticles.com

Tel: 1-631-624-4882 Email: info@cdparticles.org Address: 451

Fax: 1-631-938-8221 Ramsey Road, Shirley, NY 11967, USA

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