Analytik Jena LOOXSTER User manual

Analytik Jena AG | Life Science | Konrad-Zuse-Straße 1 | 07745 Jena / Deutschland | www.bio.analytik-jena.de
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LOOXSTER
®
Enrichment Kit
Instructions for use
englis
h
For research use only

LOOXSTER
®
Enrichment Kit 11/2013 2
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850-203-001-0010
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10
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Manufacturer:
Analytik Jena AG
Konrad-Zuse-Straße 1
07745 Jena/Germany
Phone [phone] / 77-94 00
Fax [phone]
www.bio.analytik-jena.com
[e-mail] 

LOOXSTER
®
Enrichment Kit 11/2013 3
LOOXSTER
®
Enrichment Kit Components
Storage and Shelf life
LOOXSTER
®
Magnetic Particles are packed in a resealable flat bag with clip made of
aluminium barrier film A 20T and should be stored at 2-8°C. All other components can
be stored at room temperature (15-30°C). Under these conditions a shelf life of
6 months is guaranteed.
Upon reconstitution LOOXSTER
®
Magnetic Particles should be kept at 2-8°C. Under
these conditions LOOXSTER
®
Magnetic Particles retain their performance for at least
1 week.
Product Use Limitations
LOOXSTER
®
Enrichment is a preanalytical system for enrichment of bacterial and
fungal DNA in a DNA isolate containing predominant amounts of eukaryotic DNA (cells,
tissue or body fluids).
LOOXSTER
®
Enrichment is intended for molecular biology applications. The product is
not intended for the diagnosis, prevention, or treatment of a disease.
Komponent 10 Isolations Symbol
LOOXSTER
®
Magnetic Particles 5 screwed cap tubes with lyophilisate LMP
LOOXSTER
®
Binding Buffer 1 x 8 ml LBB
LOOXSTER
®
Wash Buffer 1 x 22 ml LWB
LOOXSTER
®
Elution Buffer 1 x 6 ml LEB
Water 5 x 1.75 ml W
Tubes 2.0 ml 30 items TUB2
Desalting Spin Columns 10 items DSC
Collection Tubes 20 items CT
Desalting Binding Buffer 1 x 6 ml DBB
Desalting Wash Buffer 1 x 8 ml DWB
Desalting Elution Buffer 1 x 500 µl DEB
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Blue labelled components:storage at 2-8°C)
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All other components: storage at room temperature (15-30°C)

LOOXSTER
®
Enrichment Kit 11/2013 4
Product Warranty
We guarantee the performance of the product in the manner described in our product
information. The user must determine the suitability of the product for its particular use.
If the product does not meet your expectations, due to any reason other than misuse,
please contact us.
We reserve the right to change, alter, or modify any product in order to enhance its
performance and design.
Safety Information
When working with chemicals, always wear a suitable lab coat, disposable gloves and
protective goggles.
The following Hazard (H) and Precautionary Statements (P) apply to components of the
LOOXSTER
®
Enrichment Kit:
• Desalting Binding Buffer DBB contains guanidine hydrochloride: harmful, irritant.
Hazard and precautionary Statements: H302-H319-H315; P305+P351+P338-
P302+P352 and isopropanol: flammable, irritant. Hazard and precautionary
Statements: H225-H319-H336; P210-P233-P305+P351+P338.
• Desalting Wash Buffer DWB contains ethanol: flammable. Hazard and precautionary
Statements: H225; P210.
H225: Highly flammable liquid and vapour. H302: Harmful if swallowed. H315: Causes skin irritation.
H319: Causes serious eye irritation. H336: May cause drowsiness or dizziness.
P210: Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No
smoking. P233: Keep container tightly closed. P305+P351+P338: IF IN EYES: Rinse cautiously with
water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
P302+P352: IF ON SKIN: Wash with plenty of soap and water.
Precaution
• Please read the manual carefully prior to use LOOXSTER
®
Enrichment Kit. In case of
questions or unclarity, please contact us.
• Do not use reagents after date of expiry.
• Reagents are ready to use. Please do not modify reagents. Modification of reagents
may affect the product performance negatively.
• Avoid contaminations by using filter pipet tips.
• Use a new filter pipet tip for every sample.

LOOXSTER
®
Enrichment Kit 11/2013 5
Principle and Applications
The enrichment effect of LOOXSTER
®
is achieved by means of the specific affinity of
the LOOXSTER
®
-protein, a derivative of the human CGBP-protein, for non-methylated
CpG-dinucleotides. Bacterial and fungal genomic DNA contains unmethylated CpG-
dinucleotides or shows only a low level of CpG methylation. On the other hand, it was
shown that in mammals between 60% and 90% of all CpG-dinucleotides are methylated.
For bacterial and fungal DNA enrichment, DNA-extracts containing a mixture of
methylated host-DNA and minute amounts of double stranded genomic DNA from
bacteria or fungi, were incubated under stringent buffer conditions with
LOOXSTER
®
-protein decorated paramagnetic particles. Under these conditions the
CXXC-domain of the LOOXSTER
®
-protein forms a stable complex with non-methylated
CpG-dinucleotides (Dissociation constant: Kd ≈2.7µM
1
). Unbound methylated DNA can
be removed from the sample by means of stringent washing step. Subsequently, the
protein-DNA complex is solved in presence of an elution buffer and the enriched
bacterial or fungal DNA can be harvested from the LOOXSTER
®
-particles. After
performing a final solid-phase extraction step the desalted and concentrated bacterial or
fungal DNA can be applied to downstream analytical and preparative applications.
All components have been treated to reduce the risk of contaminations with bacterial
and fungal DNA.
Schematic representation of the
LOOXSTER
®
-principle: Total-DNA
consisting of host DNA and minute
amounts of bacterial or fungal was
isolated from human whole-blood
and applied to LOOXSTER
®
.
The specific affinity of LOOXSTER
®
for DNA molecules originating from
bacteria and fungi leads to their
enrichment relative to the
background of host DNA.

LOOXSTER
®
Enrichment Kit 11/2013 6
Sample Requirements
The function of LOOXSTER
®
relies on a specific protein-DNA-interaction. Therefore
LOOXSTER
®
should be applied to DNA-isolates that proved to be free of proteases and
denaturing or chaotropic agents (detergents, guanidine isothiocyanat, guanidine-
hydrochloride etc.) as well as chelating agents (EDTA).
In order to ensure sample requirements it is recommended to precipitate DNA-isolates
prior to application to LOOXSTER
®
Enrichment. Precipitated DNA should be rehydrated
with water.
It is recommended not to exceed 300 µg for the total DNA content present in a single
sample applied to LOOXSTER
®
Enrichment.
The DNA-binding capacity of 175 µl of reconstituted LOOXSTER
®
Magnetic P articles is
≈3.0 µg.
Essential Instrumentation
The LOOXSTER
®
Enrichment Kit contains coated paramagnetic particles. Processing of
paramagnetic particles requires a strong magnetic separator adapted to 1.5 ml and
2.0 ml reaction tubes. We recommend Magnetrack small; Analytik Jena, cat. No. 845-
MG-2000002.
For application of LOOXSTER
®
Enrichment usual laboratory instrumentation is required.
Instrument Required Specifications Manufacturer
Vortex Mixer - variable
Micro Centrifuge Rotor for 1,5 ml and 2 ml reaction tubes
(Force ca. 16.000 x g) variable
Thermomixer Thermoblock for 1.5 ml and 2.0 ml reaction
tubes variable

LOOXSTER
®
Enrichment Kit 11/2013 7
LOOXSTER
®
Enrichment Protocol
Reconstitution of lyophilized LOOXSTER
®
-Magnetic Particles
One screwed cap tube contains lyophilized LOOXSTER
®
Magnetic Particles for two
separate LOOXSTER
®
-applications. Reconstitution results in 400 µl of ready-to use
LOOXSTER
®
Magnetic Particles. If only one LOOXSTER
®
enrichment application is
carried out, the remaining volume of LOOXSTER
®
Magnetic Particles can be stored at
4-8°C for at least one week without affecting its performance.
1.
Open Screwed cap tube with lyophilized LOOXSTER
®
Magnetic Particles LMP
and add 400 µl of Water W.
Reconstitute LOOXSTER
®
Magnetic Particles LMP for 30 min using the
Thermomixer (25°C, 600 rpm).
Resuspend the reconstituted LOOXSTER
®
Magnetic Particles LMP
immediately prior to application by inverting the tube several times.
LMP
W

LOOXSTER
®
Enrichment Kit 11/2013 8
LOOXSTER
®
enrichment
1.
Adjust DNA sample using Water W to a volume of 550 µl, pipet the sample into
a Tube 2.0 ml TUB2 and add 550 µl of LOOXSTER
®
Binding Buffer LBB.
Add 175 µl of reconstituted LOOXSTER
®
Magnetic Particles LMP (carefully
resuspended) and mix the preparation by inverting the tube several times.
Incubate the preparation for 5 min in the Thermomixer (25°C, 800 rpm).
W
TUB2
LBB
LMP
2.
Place preparation in the magnetic separator and wait for 30-60 sec. Ensure
complete separation of LOOXSTER
®
Magnetic Particles LMP by carefully
inverting the Tube 2.0 ml TUB2 several times while being placed in the
magnetic separator.
Remove the particle-free supernatant from the LOOXSTER
®
Magnetic
Particles LMP.
Pipet 1 ml of LOOXSTER
®
Wash Buffer LWB into the same Tube 2.0 ml
TUB2 and close the lid. Remove tube from the magnet separator and carefully
mix the preparation by inverting the tube several times.
Place the tube in the Thermomixer and incubate preparation for 3 min at 25°C
and 800 rpm.
LWB
3.
Place preparation in the magnetic separator and wait for 30-60 sec. Ensure
complete separation of LOOXSTER
®
Magnetic Particles LMP by carefully
inverting the Tube 2.0 ml TUB2 several times while being placed in the
magnetic separator.
Remove the particle-free supernatant from the LOOXSTER
®
Magnetic
Particles LMP.
Pipet 400 µl of LOOXSTER
®
Elution Buffer LEB into the same Tube 2.0 ml
TUB2 and close the lid. Remove tube from the magnet separator and carefully
mix the preparation by inverting the tube several times.
Place the tube in the Thermomixer and incubate preparation for 3 min at 25°C
and 800 rpm.
LEB
4.
Place preparation in the magnetic separator and wait for 30-60 sec. Ensure
complete separation of LOOXSTER
®
Magnetic Particles LMP by carefully
inverting the Tube 2.0 ml TUB2 several times while being placed in the
magnetic separator.
Remove the particle-free supernatant (LOOXSTER
®
eluate) from the
LOOXSTER
®
Magnetic Particles LMP and transfer to a new Tube 2.0 ml
TUB2.
TUB2

LOOXSTER
®
Enrichment Kit 11/2013 9
Concentration and Desalting
1.
Add 400 µl Desalting Binding Buffer DBB to the Tube 2.0 ml TUB2
containing the LOOXSTER
®
eluate, close lid and vortex briefly.
Place Desalting Spin Column DSC into a Collection Tube CT and apply the
diluted LOOXSTER eluate completely to the Desalting Spin Column DSC.
Close lid and centrifuge Spin Column DSC for 30 sec at 13.000 – 16.000 x g.
DBB
DSC
CT
2.
Remove flow-through and place Desalting Spin Column DSC into the same
Collection Tube CT.
Apply 300 µl of Desalting Wash Buffer DWB to the Desalting Spin Column
DSC.
Close lid and centrifuge Spin Column DSC for 30 sec at 13.000 – 16.000 x g.
DWB
3.
Remove flow-through and place Desalting Spin Column DSC into the same
Collection Tube CT.
Apply 300 µl of Desalting Wash Buffer DWB to the Desalting Spin Column
DSC again.
Close lid and centrifuge Spin Column DSC for 30 sec at 13.000 – 16.000 x g.
DWB
4.
Remove flow-through and place Desalting Spin Column DSC into a new
Collection Tube CT.
Centrifuge Spin Column DSC for 30 sec at 13.000 – 16.000 x g.
CT
5.
Discard Collection Tube CT.
Place Desalting Spin Column DSC into a new Tube 2.0 ml TUB2.
Apply 40 µl of Desalting Elution Buffer DEB to the Desalting Spin Column
DSC. Close lid and incubate for 2 min at RT.
Centrifuge closed Spin Column DSC for 30 sec at 13.000 – 16.000 x g.
TUB2
DEB
6.
Remove Desalting Spin Column DSC from the Tube 2.0 ml TUB2.
Close lid and vortex desalted and concentrated LOOXSTER
®
eluate briefly.

LOOXSTER
®
Enrichment Kit 11/2013
10
Literature
[1] Chao Xu, Chuanbing Bian, Robert Lam, Aiping Dong & Jinrong Min. The structural
basis for selective binding of non-methylated CpG islands by the CFP1 CXXC
domain. Nat. Commun. 2011;2:227.

LOOXSTER
®
Enrichment Kit 11/2013
11
Order and Service
Product Content Order Nr. Preparations
LOOXSTER
®
Enrichment Kit
DNA-Enrichment Kit
(for bacterial and fungal DNA) 850-203-001-0010 10
Technical Support
If you have any questions or experience any difficulties regarding LOOXSTER
®
,
please do not hesitate to contact us for technical support.
You can contact us by email, phone, fax or post:

Analytik Jena AG
Life Science
Konrad-Zuse-Straße 1
07745 Jena/Germany
Tel.: [phone]
Fax: [phone]
E-Mail: [e-mail]


LOOXSTER
®
Enrichment Kit 11/2013
12

203_MA_1_11/13_EN
Symbol Explanation
Use by
Verwendbar bis
Utiliser jusqu au
Utilizzare entro
Fecha de caducidad
Catalogue number
Bestellnummer
Référence du catalogue
Numero di catalogo
Número de catálogo
Batch code
Chargenbezeichnung
Code du lot
Codice lotto
Codigo de lote
Temperature limitation
Zulässiger Temperatubereich
Limites de température
Limiti di temperatura
Limite de temperatura
Consult instructions for use
Gebrauchsanweisung beachten
Consulter le mode d emploi
Consultare le istruzioni per l uso
Consulte las instrucciones de uso
Manufacturer
Hersteller
Fabricant
Fabbricante
Productor
For research use only
Ausschließlich zu Forschungszwecken
Seulement pour rechérche
Solo per uso di Ricerca
Sólo para uso en investigación
Concentrate
Konzentrat
Concentré
Concentrato
Concentrar
Lyophilized
Lyophilisat
Lyophilisé
Liofilizzato
Liofilizado
Keep away from heat or direct sunlight
Vor Hitze und Sonneneinstrahl ung schützen
Garder à l abri de la chaleur et de toute exposition lumineuse
Non esporre ai raggi solare
Mauténgase alejado del calor o la luz solar directa
Containes sufficient for <n> tests
Ausreichend für "n" Ansätze
Contenu suffisant pour "n" tests
Contenuto sufficiente per "n" saggi
Contenido suficiente para <n> ensayos