Download Omni-Tube PCR, PCR Reaction,Digest, Ligation, and the 3

Omni-Tube PCR, PCR Reaction,Digest,
Ligation, and the 3 accompanying purification
steps in the same tube
T
he Omni-Tube PCR system is designed and optimized for performing PCR, digestion, ligation,
and purification all in a single tube. No extra tubes,
spin-columns, filter plates, silica membrane, magnetic
beads, vacuum or filtration steps are needed for purification, and no transfer of reaction mixtures to different
tubes required when proceeding to other downstream
applications. Based on Solid Surface Reversible Binding (SSRB) technology, the Omni Tube utilizes plastics coated with proprietary high efficiency-binders
acting to selectively capture and efficiently bind DNA
fragments (e.g. PCR products, digestions, ligations
etc...) from reaction mixtures. In the presence of Binding Buffer, PCR amplicons, or other types of DNA, will
specifically interact with the high efficiency-binders
and bind to the wells while primers, fluorescent dyes,
nucleotides, and other contaminants will remain in solution.
Box 1 | Contents
Kit Contents
ABP-PP-OMNIPCR25
ABP-PP-OMNIPCR50
# of
Purifications
25
50
25
50
Binding Buffer
1.5 mL
3.0 mL
Washing Buffer
3.5 mL
7.0 mL
Elution Buffer
2.5 mL
5.0 mL
# of Omni-Tubes
Additional Materials Needed
A
fter washing the tube to remove unbound material,
the purified DNA products can easily be eluted in
10 mM Tris Elution Buffer or water. The purified DNA
products can be processed directly in the binding tube
for downstream applications such as restriction enzyme digestion, cloning, SNP analysis, and sequencing without transfer to another tube. Everything from
PCR down to transformation, with purification steps in
between, performed in one Omni Tube!
Features
♦ Fast to perform: Quick 15 minute purification protocol.
♦Easy to handle: Several successive steps in a workflow can be performed in the same tube, e.g. PCR,
PCR purification, and a downstream application such
as ligation, endonuclease digestion, or sequencing, to
mention just a few, without a need for sample transfer. Since no silica membrane is needed for binding,
multiple steps of filtration in the sample binding and
washing steps associated with conventional silica
membrane methods are eliminated, which makes the
whole procedure very easy to perform.
♦ Reliable quality: Consistent performance, reliable cross-contamination prevention procedure and
unique solid-surface capture technology ensure high
reproducibility and maximum recovery of high-quality
DNA without contamination.
•96-100 % ethanol
•Isopropanol
•Benchtop Centrifuge
Box 2 | All in 1 Omni-Tube Overview
Proceed to transformation or
other downstream applications.
Purify ligation
Set up PCR Cocktail
in Omni-Tube
Run PCR using
same Omni-Tube
Surface Bind technology
allows for purification of PCR
product in same
Omni-Tube.
Purify digest in
same Omni-Tube
using same
protocol.
Proceed to digestion
reaction (if necessary)
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Ligation
(if necessary)
General Precautions
♦ This kit is for research use only. All due care
and attention should be exercised in the handling
of the kits.
♦ Wear a laboratory coat, disposable gloves,
and eye protection when handling reagents and
tubes. Avoid ingestion and inhalation of reagents.
In case of contact, wash thoroughly with water.
See Material Safety Data Sheets (MSDS) for
emergency procedures in case of accidental contact or ingestion. MSDS information is available
upon request.
♦ Always use proper aseptic techniques to avoid
nuclease contamination when working with DNA.
Use only sterile, new pipette tips to prevent cross
contamination.
Preparation
WB2: Add 100 % ethanol to Washing Buffer II
(WB2) and mix well. (Mark bottle that ethanol has
been added).
♦ Store at room temperature and use WB2 containing ethanol within six (6) months.
KB5: Prepare fresh working Binding Buffer
(KB5) prior to performing PCR product purification based on the number of samples processed.
To make working Binding Buffer (KB5), mix 10 μl
of Binding Buffer (KB5) with 40 μl 100 % Isopropanol and mix well. For working Binding Buffer
(KB5), dispense 20 μl of Binding Buffer containing Isopropanol per 10-μl PCR solution.
♦ Discard the unused Binding Buffer at the end
of the day.
♦ The kits are designed to purify PCR fragment
sizes of greater than 60 base pairs from PCR reactions. After PCR,cool the reaction to room temperature before starting the purification.
Protocols
♦ The protocol below is for the purification of PCR DNA amplicons from 50-μl PCR using the Omni-Tube PCR system.
The purification procedure may be scaled from 10 – 100
μl by proportionately adjusting all reagents throughout the
procedure
Binding DNA Products
1. PCR can be performed directly in the OmniTube. If PCR was performed in a different tube,
transfer 50 μl of PCR mixture from the PCR tube
to the Omni-Tube
2. Add 100 μl of working Binding Buffer (KB5)
containing isopropanol to each sample well. Mix
well by pipetting solution up and down 7 – 8 times.
3. Centrifuge at >2,250 x g for 1 min.
4. Remove the liquid from the SurfaceBind tube.
Choose one of the methods listed below to remove the liquid:
(A) Decant the solution by quickly flipping the
tube over a waste container and shaking briskly,
then place the inverted plate on a stack of clean
absorbent paper, such as Kimwipe® or paper
towels, and tap the tube on the clean paper 3 – 4
times to remove as much liquid as possible;
(B) Remove the solution by aspirating the solution from each well with a multi-channel pipettor.
Be sure not to scrape the walls of the wells during
aspiration as the products are bound to the walls.
Washing DNA Products
1. Add 150 μl Wash Buffer containing ethanol to
each well. Mix by pipetting solution up and down
2 – 3 times and incubate the tube for 30 seconds
at room temperature.
2. Remove the Wash Buffer from the Omni-Tube
using one of the methods suggested in step 4
above.
3. (Optional) Repeat steps 1 and 2 above for a
total of two washes. *One wash is sufficient for
most applications
4. After the final wash blot the inverted tube dry
on a piece of clean absorbent paper 3 – 4 times
and air-dry the tube for 5 – 8 minutes to remove
any residual liquid.
Eluting DNA Products
♦ PCR amplicons attached to the wall of the Binding Plate
can be analyzed directly in the well they were purified in
by restriction enzyme digestion, primer extension, SNP detection and sequencing using a 25 – 50 μl reaction volume
without elution. If the DNA is to be eluted, use the procedure
below
1. Add 50 μL Elution Buffer (EB1) into the OmniTube. (If higher concentration is preferred, add
25 μL Elution Buffer (EB1) and use Option 1 to
elute PCR product).
2. Choose one of the following Options:
(Option 1): (Elute with vortex): Vortex the tube
for 30 seconds. Centrifuge the tube at maximum
speed for 1 minute to collect all liquid at the bottom of each well.
(Option 2): (Elute without vortex): Pipette the solution up and down 5 – 7 times, seal the tube and
incubate for 60 seconds at room temperature.
3. The eluted PCR sample can be used immediately for downstream application. Alternatively,
the eluted PCR sample may be stored in the
sealed plate at 4 º C for short-term storage or
–20 º C for long-term storage.
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Box 2 | Troubleshooting
Problem
Cause
Solution
No PCR product
PCR failure
Be sure to add all components of the PCR
mixture. Check PCR positive control.
Low PCR yield
Check yield of unpurified PCR product on
agarose gel.
Short centrifugation
If lower speed centrifuge is used, extend the
spin time.
Low yield of
product
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