Macherey-Nagel NucleoBond Xtra Midi User Manual
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Macherey-Nagel NucleoBond Xtra Midi User Manual

Plasmid dna purification
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MACHEREY-NAGEL
MACHEREY-NAGEL
MACHEREY-NAGEL
Plasmid DNA
Purification
User Manual
NucleoBond
NucleoBond
NucleoBond
NucleoBond
January 2008/Rev. 04
®
Xtra Midi
®
Xtra Maxi
®
Xtra Midi Plus
®
Xtra Maxi Plus
MN

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Summary of Contents for Macherey-Nagel NucleoBond Xtra Midi

  • Page 1 Plasmid DNA Purification User Manual ® NucleoBond Xtra Midi ® NucleoBond Xtra Maxi ® NucleoBond Xtra Midi Plus ® NucleoBond Xtra Maxi Plus January 2008/Rev. 04 MACHEREY-NAGEL MACHEREY-NAGEL MACHEREY-NAGEL...
  • Page 2 TE buffer 200-800 µl volume of TE buffer 400-1000 µl MACHEREY-NAGEL GmbH & Co. KG • Neumann-Neander-Str. 6-8 • D-52355 Düren • Germany Tel.: +49 (0) 24 21 969 270 • Fax: +49 (0) 24 21 969 279 • e-mail: tech-bio@mn-net.com...

  • Page 3: Table Of Contents

    ® NucleoBond Finalizers - English 7.4 High-copy plasmid purification (Midi, Maxi) - German 7.5 Low-copy plasmid purification (Midi, Maxi) - German ® 7.6 Concentration of NucleoBond Xtra eluates with the ® NucleoBond Finalizers – German MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 4 7.8 Low-copy plasmid purification (Midi, Maxi) – French ® 7.9 Concentration of NucleoBond Xtra eluates with the ® NucleoBond Finalizers – French 8 Appendix 8.1 Troubleshooting 8.2 Ordering information 8.3 Product use restriction / warranty MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 5: Components

    NucleoBond Xtra Midi Column Filters ® NucleoBond Finalizers 30 ml Syringes 1 ml Syringes Buffer TRIS 15 ml 75 ml Plastic Washers User Manual For preparation of working solutions and storage conditions see section 5. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 6 Maxi Column Fil- ters ® NucleoBond Finalizers Large 30 ml Syringes 1 ml Syringes Buffer TRIS 15 ml 75 ml Plastic Washers User Manual For preparation of working solutions and storage conditions see section 5. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 7: Reagents And Equipment To Be Supplied By User

    • 70% ethanol (room-temperatured) • Buffer for reconstitution of DNA, e.g. TE buffer or sterile H O (not necessary for NucleoBond Xtra Midi/Maxi Plus kits) Equipment: • Standard microbiological equipment for growing and harvesting bacteria (e.g. inoculating loop, culture tubes and flasks, 37°C shaking incubator, and centri- fuge with rotor and tubes or bottles for harvesting cells) •...

  • Page 8: Kit Specifications

    DNA can be eluted with a concentration up to 3 µg/µl (see section 4.11, Table 4 and 5 for dependence of concentration on elution volume). ® • All NucleoBond Finalizers are resistant to organic solvents such as alcohol, chloroform, and phenol and are free of endotoxins. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 9: About This User Manual

    8 ml of Buffer LYS when per- forming a Midi prep and in 12 ml for a Maxi prep. Follow the handling instructions exactly and note the given hints for protocol alterations. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 10: Nucleobond

    Xtra anion-exchange columns ® NucleoBond Xtra is a silica-based anion-exchange resin, developed by MACHEREY-NAGEL and covered under European Patent EP 0 496 822. It is devel- oped for routine separation of different classes of nucleic acids like oligonucleotides, RNA, and plasmids. ®...
  • Page 11 The more interactions a nucleic acid can form between the phosphate backbone and the positively charged resin the later it is eluted with increasing salt concentration. Large nu- cleic acids carry more charges than short ones, double stranded DNA more than single stranded RNA. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 12: Growth Of Bacterial Cultures

    Maniatis T, Fritsch EF, Sambrook J: Molecular cloning. A laboratory manual, Cold Spring Harbor, Cold Spring, New York 1982. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 13: Culture Volume For High-Copy Plasmids

    300 ml 200 ml 150 ml 120 ml Table 2 shows recommended ODVs and the corresponding pairs of OD and cul- ture volume that can be easily handled using the standard kit protocol lysis buffer volumes. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 14: Culture Volume For Low-Copy Plasmids

    Recommended culture volume for Pellet ® NucleoBond Rec. Xtra kit weight Midi 1.50 g 400 ml 200 ml 133 ml 100 ml 80 ml Maxi 4.50 g 2400 1200 ml 600 ml 400 ml 300 ml 240 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 15: Cell Lysis

    Therefore, resuspend the cell pellet in Buffer RES containing 2 mg/ml lysozyme and incubate at 37°C for 30 minutes. Proceed then with the lysis procedure according to ® the NucleoBond Xtra standard protocol. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 16: Setup Of Nucleobond

    Columns can also be placed in the NucleoBond Rack Large (Cat. No. 740563). Figure 3 ® ® Setup of NucleoBond Xtra Midi/Maxi Columns with the NucleoBond Xtra Combi Rack A: Setup for clarification, loading, and first washing step, B: Setup for elution. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 17: Filtration And Loading Of The Lysate

    ® It is essential to wash the NucleoBond Xtra column without filter for a second time with Wash Buffer WASH. This ensures highest yields with best achievable purity. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 18: Elution And Concentration Of Plasmid Dna

    Figure 4 and 5 illustrate exemplarily how DNA recovery and final DNA concentration ® depend on the buffer volume which is used for elution of DNA from the NucleoBond ® Finalizer and NucleoBond Finalizer Large, respectively. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 19 0.1 µg/µl 0.1 µg/µl 0.1 µg/µl 30 % 75 % 85 % 90 % 90 % 90 % 50 µg 0.3 µg/µl 0.2 µg/µl 0.1 µg/µl 0.1 µg/µl 0.1 µg/µl <0.1 µg/µl DNA recovery DNA concentration MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 20 0.8 µg/µl 0.6 µg/µl 0.5 µg/µl 15 % 45 % 70 % 80 % 85 % 90 % 100 µg 0.4 µg/µl 0.3 µg/µl 0.2 µg/µl 0.1 µg/µl 0.1 µg/µl 0.1 µg/µl DNA recovery DNA concentration MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 21: Determination Of Dna Yield And Quality

    4°C. Note that the eluate should be warmed up to room temperature before precipitating the DNA to avoid co-precipitation of salt. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 22: Storage Conditions And Preparation Of Working Solutions

    Ref. 740410.100 contains 2 x 30 mg of RNase A. Make sure to dissolve RNase A of both vials, each in 1 ml of Buffer RES, and transfer the solution back into the bottle containing Buffer RES. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 23: Safety Instructions - Risk And Safety Phrases

    Art. 12 and German GefStoffV § 42 and TRGS 200 7.1) Label not necessary, if quantity below 25 g or ml (according to 67/548/EEC Art. 25, 1999/45/EC Art. 12 and German GefStoffV § 42 and TRGS 200 7.1) MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 24: Nucleobond

    If the culture volume is more than double the recommended culture volume, it is advantageous to use a centrifuge for the lysate clarifi- ® cation in step 8 rather than the NucleoBond Xtra column filters. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 25 Apply the buffer onto the rim of the column filter as shown in the picture. Allow the column to empty by gravity flow and make sure to wet the entire filter. The column does not run dry. 12 ml 25 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 26 Al- low the column to empty by gravity flow. Note: You may want to save all or part of the flow-through for analysis (see section 8.1). MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 27 Finalizer ® Large (NucleoBond Xtra Maxi Plus). Optional: Determine plasmid yield by UV spectrophotometry in order to adjust desired concentration of DNA in step 15 and calculate the recovery after precipitation. 5 ml 15 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 28 10-60 min (3D- shaker). Determine plasmid yield by UV spectrophotometry. Confirm plasmid integrity by agarose gel electrophoresis (see section 4.12). MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 29: Low-Copy Plasmid Purification (Midi, Maxi)

    Additional lysis buffer ® volumes might have to be ordered separately (see ordering information for NucleoBond Xtra Buffer Set I, section 8.2). Use a centrifuge for the lysate clarification rather than the ® NucleoBond Xtra column filters. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 30 Apply the buffer onto the rim of the column filter as shown in the picture. Allow the column to empty by gravity flow and make sure to wet the entire filter. The column does not run dry. 12 ml 25 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 31 An incubation of the lysate is not necessary. Note: Increase NEU buffer volume proportionally if more than the recommended cell mass is used (see section 4.6 for information on optimal cell lysis). 16 ml 24 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 32: Concentration Of Nucleobond Nucleobond

    Finalizer to the syringe outlet. Fill 70 % ethanol (not supplied with the kit) into the syringe, insert the plunger ® and press the ethanol slowly through the NucleoBond Finalizer. Discard the ethanol. 2 ml 5 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 33 Transfer the first eluate back into the syringe and elute into the same collecting tube a second time. load first eluate load first eluate completely completely Determination of yield Determine plasmid yield by UV spectroscopy and confirm plasmid integrity by agarose gel electrophoresis (see section 4.12). MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 34: High-Copy Plasmid Purification (Midi, Maxi)

    (s. Bestellinformation für das NucleoBond Xtra Buffer Set I, Kapitel 8.2). Falls das Kulturvolumen mehr als doppelt so hoch ist wie das empfohlene, ® verwenden Sie in Schritt 8 zur Lysatklärung eine Zentrifuge anstelle des NucleoBond Xtra Filters. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 35 Inkubieren Sie die Mischung für 5 min bei Raumtemperatur (20-25°C). Hinweis: Erhöhen Sie das Volumen des Puffers LYS proportional falls mehr als die emp- fohlene Zellmasse eingesetzt wird (für Informationen zur optimalen Zelllyse siehe Kapitel 4.6). 8 ml 12 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 36 Eine Inkubation des Lysates ist nicht not- wendig. Hinweis: Hinweis: Erhöhen Sie das Volumen des Puffers NEU proportional falls mehr als die empfohlene Zellmasse eingesetzt wird (für Informationen zur optimalen Zelllyse siehe Kapitel 4.6). 8 ml 12 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 37 Filter verbliebene Lysat ausgewa- schen wird. Wird dieser Schritt weggelassen oder der Puffer direkt in den Säulenfilter statt auf den Rand gege- ben, kann dies zu reduzierter Plasmidausbeute führen. 5 ml 15 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 38 Vortexen und lassen Sie die Mischung für 2 Minuten stehen. Zentrifugieren Sie bei 5,000 x g für 15 min bei Raumtemperatur, vorzugs- weise bei 15,000 x g für 30 min und 4°C. Dekantieren Sie vorsichtig den Über- stand. 3.5 ml 10.5 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 39 Auf- und Abpipettieren oder unter gleichmäßigem Schütteln in einem ausreichenden Volumen Puffer für 10-60 Min (3D-Schüttler) erfolgen. Bestimmen Sie photometrisch die Plasmidausbeute und überprüfen Sie die Plas- midintegrität mittels Agarosegel-Elektrophorese (siehe Kapitel 4.12). MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 40: Low-Copy Plasmid Purification (Midi, Maxi)

    Kapitel 4.5) und in Schritt 3 entschieden werden, wie viele Zellen für die Prä- paration eingesetzt werden. Das unten aufgeführte Volumen der Übernachtkultur ist be- rechnet für eine finale OD von 4 (siehe auch Kapitel 4.5). 200 ml 600 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 41 Hinweis: Erhöhen Sie das Volumen des Puffers RES proportional falls mehr als die empfohlene Zellmasse eingesetzt wird (für Informationen zur optimalen Zelllyse siehe Kapitel 4.6, für schwer zu lysierende Bakterienstämme siehe Kapitel 4.7). 16 ml 24 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 42 Abbildung rechts gezeigt. Lassen Sie die Flüs- sigkeit vollständig durch die Säule laufen und stellen Sie ® sicher, dass der NucleoBond Xtra Filter komplett benetzt ist. Die Säulen laufen nicht trocken. 12 ml 25 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 43 Eine Inkubation des Lysates ist nicht notwendig. Hinweis: Erhöhen Sie das Volumen des Puffers NEU proportional falls mehr als die empfohlene Zellmasse eingesetzt wird (für Informationen zur optimalen Zelllyse siehe Kapitel 4.6). 16 ml 24 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 44: Concentration Of Nucleobond Nucleobond

    Füllen Sie 70 %iges Ethanol (nicht im Lieferumfang enthalten) in die Spritze, setzen Sie den Kolben ein und drücken Sie das Ethanol langsam durch den ® NucleoBond Finalizer. Verwerfen Sie das Ethanol. 2 ml 5 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 45 Kolben heraus und befestigen den NucleoBond Finalizer wieder am Auslass der Spritze. Überführen Sie das erste Eluat zurück in die Spritze und eluieren ein zweites Mal in dasselbe Auffanggefäß. erstes Eluat erstes Eluat vollständig laden vollständig laden MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 46 ® NucleoBond Xtra Midi/Maxi - German ® ® Midi - NucleoBond Maxi - NucleoBond Finalizer Finalizer Large Bestimmung der Plasmid-Ausbeute Bestimmen Sie photometrisch die Plasmidausbeute und überprüfen Sie die Plasmidintegrität mittels Agarosegel-Elektrophorese (siehe Kapitel 4.12). MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 47: High-Copy Plasmid Purification (Midi, Maxi) - French

    Xtra Buffer Set I, chapitre 8.2). Si le volume de culture est plus du double du volume recommandé, il est plus judicieux de centrifuger pour clarifier le ® lysat (étape 8), plutôt que d’utiliser le filtre NucleoBond Xtra. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 48 Remarque : Augmentez proportionnellement le volume du tampon LYS si vous avez utilisé une masse cellulaire supérieure à celle recommandée (voir section 4.6 pour plus d’informations sur la lyse optimale des cellules). 8 ml 12 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 49 Remarque : Augmentez proportionnellement le volume du tampon NEU si vous avez utilisé une masse cellulaire supérieure à celle recommandée (voir section 4.6 pour des informations sur la lyse optimale des cellules). 8 ml 12 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 50 Omettre cette étape ou déposer simplement le tampon dans le filtre réduit le rendement d’ADN plasmidique. 5 ml 15 ml 10 Eliminer le filtre ® Retirez le filtre NucleoBond Xtra ou éliminez le en retournant la colonne. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 51 élué. Vortexez et laissez le mélange reposer pendant 2 minutes. Centrifugez à 5,000 x g pendant 15 min à température ambiante, de préférence à 15,000 x g pendant 30 min à 4°C. Eliminez précautionneusement le surnageant. 3.5 ml 10.5 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 52 - refoulement ou par rotation constante dans un volume approprié de tampon pendant 10-60 min (3D-shaker). Déterminez le rendement d’ADN plasmidique par spectrophotométrie UV. Confirmez l’intégrité du plasmide par électrophorèse sur gel d’agarose (voir la section 4.12). MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 53: Low-Copy Plasmid Purification (Midi, Maxi) - French

    à l’étape 3 de la quantité de cellules à utiliser pour la préparation. Le volume mentionné ci-après de la culture overnight est calculé pour la valeur de l’OD de 4 (voir chapitre 4.5 comme référence). 200 ml 600 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 54 à celle recommandée (voir la section 4.6 pour plus d ‘informations sur les conditions de lyse optimales des cellules et la section 4.7 pour les souches difficiles à lyser). 16 ml 24 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 55 Déposez le tampon sur la collerette du filtre comme le montre le schéma. Laissez la colonne se vider par gravité et assurez vous que le filtre est entièrement mouillé. La colonne ne s’assèche pas. 12 ml 25 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 56 Remarque : augmentez proportionnellement le volume de tampon NEU si vous avez utilisé une masse cellulaire supérieure à celle recommandée (voir la section 4.6 pour plus d’informations sur la lyse optimale des cellules). 16 ml 24 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 57 Finalizer à la sortie de la seringue. Versez d’éthanol 70% (non fourni dans les kits) dans la seringue, insérez le ® piston, et pressez l’éthanol doucement à travers le NucleoBond Finalizer. Eliminez l‘éthanol. 2 ml 5 ml MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 58 NucleoBond Finalizer à la sortie de la seringue. Transférez le premier éluat dans la seringue et éluez dans le même tube collecteur une seconde fois. Charger complètement Charger complètement le le premier éluat premier éluat MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 59 ® ® Midi - NucleoBond Maxi - NucleoBond Finalizer Finalizer Large Détermination du rendement Déterminez le rendement d’ADN plasmidique par spectrophotométrie UV et confirmez l’intégrité du plasmide par électrophorèse sur gel d’agarose (voir section 4.12). MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 60: Appendix

    (lanes 1 and 2). It might also occur in the wash fraction but must be absent in the eluate. Genomic DNA should not be visible at all but would show up in the gel slot or right below indicating e.g. too harsh lysis conditions. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 61 I, cleared lysate, ccc, linear and oc structure of the plasmid, degraded RNA II, lysate flow-through, no plasmid DNA, but degraded RNA III, wash flow-through, no plasmid DNA or residual RNA IV, eluate, pure plasmid DNA EcoRI restriction, linearized form of plasmid MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 62 • Check plasmid content in the wash fractions (see Figure 6). Keep all buffers tightly closed. Check and adjust pH of Buffer EQU (pH 6.5) and ELU (pH 9.0) with HCl or NaOH if necessary. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 63 Lysate was mixed too vigorously or vortexed after lysis of plasmid • Invert tube for only 5 times. Do not vortex after addition of LYS. • Use larger tubes or reduce culture volumes for easier mixing. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 64 Nucleic acid did not precipitate • Check type and volumes of precipitating solvent. Make sure to use at least 0.7 volumes of isopropanol and mix thoroughly. • Centrifuge for longer periods of time at higher speed. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 65 Refer to section 4.11, Table 4 and 5 to estimate the recovery that can be expected depend- ing on elution buffer volume. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 66 • Plasmid DNA was not dried completely before redissolving. Pre- subsequent cipitate DNA again by adding 1/10 volume of 3 M NaAc pH 5.0 reactions and 0.7 volumes of isopropanol. Proceed with the precipitation protocol in this manual und dry DNA pellet completely. MACHEREY-NAGEL – 01/2008/ Rev. 04...
  • Page 67 DNA is irreversibly denatured reactions (continued) • A denatured plasmid band runs faster on the gel than the super- coiled conformation. Do not lyse the sample after addition of Buffer LYS for more than 5 minutes. MACHEREY-NAGEL – 01/2008/ Rev. 04...

  • Page 68: Ordering Information

    This MACHEREY-NAGEL product is shipped with documentation stating specifica- tions and other technical information. MACHEREY-NAGEL warrants to meet the stated specifications. MACHEREY-NAGEL´s sole obligation and the customer´s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted.
  • Page 69 Plasmid DNA Purification terms and conditions of MACHEREY-NAGEL, which are printed on the price list. Please contact us if you wish an extra copy. MACHEREY-NAGEL does not warrant against damages or defects arising in shipping and handling (transport insurance for customers excluded), or out of accident or improper or abnormal use of this product;...

This manual is also suitable for:

Nucleobond xtra maxiNucleobond xtra midi plusNucleobond xtra maxi plus740410.10740410.50740410.100 ... Show all

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