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Plasmid DNA Purification

1. Add 0 7 volumes of room temperature isopropanol not supplied with the kit Vortex well and let the mixture sit for 2 minutes e g for 5 ml NucleoBond Xtra Midi eluate add 3 5 ml isopropanol for 15 ml NucleoBond Xtra Maxi eluate add 10 5 ml isopropanol Note The NucleoBond Finalizer only holds up to 500 ug and the NucleoBond Finalizer Large is limited to 2000 L g of plasmid DNA Please check plasmid content of your eluate prior to the precipitation step by measuring A see section 4 11 Load ing more DNA might lead to clogging and complete loss of your sample 3 5 ml for 10 5 ml for 5 ml eluate 15 ml eluate 2 Load precipitate Remove the plunger from a 30 ml syringe and attach a NucleoBond Finalizer to the outlet Fill the precipitation mixture into the syringe insert the plunger and press the mixture slowly through the NucleoBond Finalizer using constant force Discard the flow through 3 Wash precipitate Remove the NucleoBond Finalizer from the syringe pull out the plunger and reattach the NucleoBond Finalizer to the syringe outlet Fill 2 ml of 70 ethanol not supplied with the kit into the syringe insert the plunger and press the ethanol slowly through the NucleoBond Finalizer Dis card the ethanol 48 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi Midi NucleoBond Maxi NucleoBond Finalizer Finalizer Large Dry fil
2. Plasmid DNA Purification User manual NucleoBond Xtra Midi NucleoBond Xtra Maxi NucleoBond Xtra Midi Plus NucleoBond Xtra Maxi Plus March 2006 Rev 01 MACHEREY NAGEL MN Protocol at a glance Rev 01 Plasmid DNA Purification NucleoBond Xtra Midi Maxi 1 Cultivate and harvest 4 500 6 000 x g bacterial cells 15 min at 4 C 2 Cell lysis high copy low copy high copy low copy Buffer RES 8 ml 16 ml 12 ml 24 ml Buffer LYS 8 ml 16 ml 12 ml 24 ml 3 Equilibration of the column together with inserted column filter Buffer EQU Buffer EQU 12 ml 25 ml 4 Neutralization Buffer NEU Buffer NEU 8 ml 16 ml 12 ml 24 ml 5 Clarification and invert the tube 3 times loading of the lysate load lysate on NucleoBond Xtra column filter lysate is simultaneously cleared and loaded onto the NucleoBond Xtra column Buffer EQU Buffer EQU 6 1 Washing 5 ml 15 ml e e 7 Discard NucleoBond discard NucleoBond Xtra discard NucleoBond Xtra Xtra column filter column filter column filter na g Buffer WASH Buffer WASH 8 2 Washing 8 ml 25 ml e 9 Elution Buffer ELU Buffer ELU 5 ml 15 ml es NucleoBond Xtra NucleoBond Xtra oBond oBond 10 Precipitation Midi Midi Plus D Isopropanol Isopropanol Isopropanol Isopropanol 3 5 ml 3 5 ml 10 5 ml 10 5 ml 15 000 x g load NucleoBond 15 000 xg load NucleoBond 30 min at 4 C Finalizer 30 min at 4 C Finalizer La
3. de tampon pendant 10 60 min 3D shaker Determinez le rendement d ADN plasmidique par spectrophotom trie UV Confirmez l int grit du plasmide par lectrophor se sur gel d agarose voir la section 4 11 MACHEREY NAGEL 03 2006 Rev 01 37 NucleoBond Xtra Midi Maxi 7 4 Low copy plasmid purification Midi Maxi English The lysis buffer volumes provided in the kit are adjusted for high copy plasmid purifi cation Therefore additional buffer has to be ordered separately for routine purifica tion of low copy plasmids see section 9 2 for ordering information Prepare a starter culture Inoculate a 3 5 ml starter culture of LB medium with a single colony picked from a freshly streaked agar plate Make sure that plate and liquid culture contain the appropriate selective antibiotic to guarantee plasmid propagation see section 4 3 for more information Shake at 37 C and 300 rpm for 8 h o aD Prepare a large overnight culture Inoculate an overnight culture by diluting the starter culture 1 1000 into the given volumes of LB medium also containing the appropriate selective antibiotic If the culture is Known to grow poorly consult section 4 5 for larger culture volumes Grow the culture overnight at 37 C and 300 rpm for 12 16 h Note To utilize the entire large binding capacity of the NucleoBond Xtra columns it is important to provide enough plasmid DNA For the standard low copy procedure the cul
4. zzxz Cell lysis Check lysis buffer LYS for precipitated SDS prior to use If a white precipitate is visible warm the buffer for several minutes at 30 40 C until precipitate is dis solved completely Cool buffer down to room temperature 20 25 C Add lysis buffer LYS to the suspension Mix gently by inverting the tube 5 times Do not vortex as this will shear and release contaminating chromosomal DNA from cellular debris into the suspension Incubate the mixture at room temperature 20 25 C for 5 min Note Increase LYS buffer volume proportionally if more than the recommended cell mass is used see section 4 6 for information on optimal cell lysis 22 Equilibration Equilibrate a NucleoBond Xtra column together with the inserted column filter with equilibration buffer EQU 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 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 7 9 zxzzp Neutralization Add neutralization buffer NEU to the suspension and immediately mix the lysate gently by inverting the tube 10 15 times Do not vortex The flask or tube used for this step should not be filled more than two thirds to allow homogeneous mixing Make sure to neutralize completely to precipitate all the protein and chromo
5. ditionally contain the NucleoBond Finalizer and NucleoBond Finalizer Large respectively These tools for a fast concentration and desalination of eluates are suitable for most plasmids and cosmids ranging from 2 50 kb with recovery efficiencies from 40 90 depending on elution volume NucleoBond Finalizer is a polypropylene syringe filter containing a special silica membrane The NucleoBond Finalizer provides a binding capacity of 500 ug whereas the NucleoBond Finalizer Large can hold up to 2000 ug plasmid DNA Due to the small dead volumes of NucleoBond Finalizers the plasmid DNA can be eluted with a concentration up to 2 0 ug ul see section 4 10 Figures 3 and 4 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 03 2006 Rev 01 7 Plasmid DNA Purification 3 About this user manual The following section 4 provides you with a detailed description of the NucleoBond Xtra purification system and important information about cell growth cell lysis and the subsequent purification steps Sections 5 and 6 inform you about storage buffer preparation and safety instructions First time users are strongly advised to read these chapters thoroughly before using this kit Experienced users can directly proceed with the purification protocols sec tion 7 or just use the Protocol a
6. ture volumes were doubled compared to the high copy vector protocol However due to a plasmid content that is 10 100 times lower this might be insufficient If you need large amounts of low copy plasmids further increase the culture volume by factor 3 5 see section 4 5 for more information and decide in step 3 how much cells to use for the preparation Harvest bacterial cells Measure the cell culture ODgo and determine the recommended culture volume Pellet the cells by centrifugation at 4 500 6 000 x g for 210 min at 4 C and dis card the supernatant completely Note It is of course possible to use larger culture volumes e g if a large amount of low copy plasmid is needed see section 4 5 for more information In this case increase RES LYS and NEU buffer volumes proportionally in steps 4 5 and 7 and use a centrifuge for the lysate clarification rather than the NucleoBond Xtra column filters 38 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 4 na Resuspension Resuspend ihe cell pellet completely in resuspension buffer RES RNase A by pipetting up and down or vortexing the cells For an efficient cell lysis it is important that no clumps remain in the suspension Note Increase RES buffer volume proportionally if more than the recommended cell mass is used see section 4 6 for information on optimal cell lysis and section 4 7 re garding difficult to lys
7. 4 6 quilibrierung quilibrieren Sie eine NucleoBond Xtra S ule zusammen mit dem eingesetzten NucleoBond Xtra Filter mit equilibration buffer EQU Geben Sie den Puffer auf den u eren Rand des Filters wie in der Abbildung rechts gezeigt Lassen Sie die Fl s sigkeit vollst ndig durch die S ule laufen und stellen Sie sicher dass der NucleoBond Xtra Filter komplett benetzt ist Die S ulen laufen nicht trocken em RES MACHEREY NAGEL 03 2006 Rev 01 43 NucleoBond Xtra Midi Maxi 7 o lt znp Neutralisation Geben Sie neutralization buffer NEU zum Lysat und mischen Sie sofort aber vorsichtig durch 10 bis 15 maliges Invertieren Vortexen Sie nicht Das fur diesen Schritt verwendete GefaB sollte nicht mehr als zwei Drittel gefullt sein um ein gleichm iges Durchmischen zu erm glichen Stellen Sie sicher dass die Neutralisation vollst ndig erfolgt ist um eine quantitative F llung von Protein und genomischer DNA zu gew hrleisten Das schleimige viskose Lysat sollte nach Zugabe des Puffers NEU d nnfl ssig werden und eine homogene Suspension mit flockigem wei em Pr zipitat ausbilden Fahren Sie sofort mit Kapitel 7 2 Schritt 8 des high copy Plasmid Protokolls fort Eine Inkubation des Lysates ist nicht notwendig Hinweis Erh hen Sie das Volumen des Puffers NEU proportional falls mehr als die empfohlene Zellmasse eingesetzt wird f r Informationen zur optimalen Zelllyse siehe Kapitel
8. Geben Sie Iysis buffer LYS zu der Suspension Mischen Sie vorsichtig durch 5 maliges Invertieren des Gef es Vortexen Sie nicht da dies zur Scherung der genomischen DNA und zu deren Freisetzung aus den Zelltrummern in die Suspension f hren kann Inkubieren Sie die Mischung f r 5 min bei Raumtemperatur 20 25 C Hinweis Erh hen Sie das Volumen des Puffers LYS proportional falls mehr als die emp fohlene Zellmasse eingesetzt wird f r Informationen zur optimalen Zelllyse siehe Kapitel 4 6 MACHEREY NAGEL 03 2006 Rev 01 27 NucleoBond Xtra Midi Maxi 6 Aquilibrierung quilibrieren Sie eine NucleoBond Xtra S ule zusammen mit dem eingesetzten NucleoBond Xtra Filter mit equi libration buffer EQU Geben Sie den Puffer auf den u eren Rand des Filters wie in der Abbildung rechts gezeigt Lassen Sie die Fl s sigkeit vollst ndig durch die S ule laufen und stellen Sie sicher da der NucleoBond Xtra Filter komplett benetzt ist Die S ulen laufen nicht trocken 9 zzzp Neutralisation Geben Sie neutralization buffer NEU zu der Suspension und mischen Sie sofort aber vorsichtig durch 10 bis 15 maliges Invertieren Vortexen Sie nicht Das f r diesen Schritt verwendete GefaB sollte nicht mehr als zwei Drittel gef llt sein um ein gleichmaBiges Durchmischen zu erm glichen Stellen Sie sicher dass die Neutralisation vollstandig ist um eine quantitative F
9. beeinflusst wer Waschen der S ule washing buffer WASH Waschen Sie die NucleoBond Xtra S ule mit washing buffer WASH Es ist wichtig den Filter vor Aufgabe des Waschpuffers zu den kann 8 ml 12 Elution Eluieren Sie die Plasmid DNA mit elution buffer ELU Fahren Sie mit Schritt 13 fort um die Isopropanol F llung gem Zentrifugati onsprotokoll durchzuf hren oder folgen Sie der Anleitung in Kapitel 8 zur Kon zentrierung und Entsalzung mittels NucleoBond Finalizer NucleoBond Xtra Midi Plus oder NucleoBond Finalizer Large NucleoBond Xtra Maxi Plus Optional Bestimmen Sie photometrisch die Plasmidausbeute um in Schritt 15 die ge w nschte DNA Konzentration einstellen und die endg ltige Ausbeute nach der Pr zipita tion bestimmen zu k nnen 13 Pr zipitation Pr zipitieren Sie die eluierte Plasmid DNA durch Zugabe von Isopropanol wobei der Alkohol Raumtemperatur haben sollte Mischen Sie sofort gr ndlich durch Vortexen und lassen Sie die Mischung f r 2 Minuten stehen Zentrifugieren Sie bei 5 000 x g f r 215 min bei s Raumtemperatur vorzugs weise bei 15 000 x g f r 30 min und 4 C Dekantieren Sie vorsichtig den ber stand 30 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 14 Waschen und Trocknen des DNA Pellets Waschen Sie das DNA Pellet mit ebenfalls Raumtemperatur warmem 70 igem Ethanol und zentr
10. column filter inserted in the NucleoBond Xtra column This ensures com plete removal of SDS precipitates Incubation of cleared lysates for longer periods of time might lead to formation of new pre cipitate If precipitate is visible it is recommended to filter or centrifuge the lysate again directly before loading it onto the Nu cleoBond Xtra column Sample lysate is too viscous Too much cell mass was used Refer to section 4 4 4 6 regard ing recommended culture volumes and lysis buffer volumes Make sure to mix well after neutralization to completely precipi tate SDS and chromosomal DNA Otherwise filtration efficiency and flow rate go down and SDS prevents DNA from binding to the column pH or salt concentrations of buffers are too high e Keep all buffers tightly closed Check and adjust pH of buffer LYS pH 6 5 and NEU pH 9 0 58 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification Problem Possible cause and suggestions Culture volumes are too large Refer to section 4 4 4 6 regarding recommended culture vol umes and larger lysis buffer volumes NucleoBond Xtra column Precipitate was not resuspended before loading nes clogs e Invert crude lysate at least 3 times directly before loading during filtra tion ERS Incomplete precipitation step Make sure to mix well after neutralization to completely precipi tate SDS and chromosomal DNA Sample is too viscous Do NOT attempt to puri
11. high due to inefficient washing Remove the NucleoBond Xtra column filter BEFORE performing the second washing step with buffer WASH Buffer WASH instead of buffer EQU was used for the first wash Buffer EQU has to be used to wash out the NucleoBond Xtra column filter to avoid protein carryover Only minimal amounts of DNA were loaded onto the column To much free binding capacity needs more extensive washing double washing step with buffer WASH White pre cipitate in elu ate NucleoBond Xtra column filter was not removed before second washing step e Spin down the white precipitate and continue with DNA precipi tation from supernatant Co precipitation of salt Check isopropanol purity and perform precipitation at room heey ace temperature 20 25 C but centrifuge at 4 C Do not let the elu an t ate drip from the column into isopropanol but add isopropanol to white instead the final eluate and mix immediately p clear and e Try resuspending the pellet in buffer WASH and reload onto the grassy same NucleoBond Xtra column Wash the column several times with buffer WASH before loading 60 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification Problem Possible cause and suggestions No nucleic acid pellet formed after precipitation Pellet was lost Handle the precipitate with care Decant solutions carefully De termine DNA yield in buffer ELU in order to calculate th
12. section 4 9 Figures 3 Midi ou 4 Maxi pour choisir le volume appropri de tampon d lution Pipetez 500 1000 ul de tampon d elution buffer TRIS 5 mM Tris HCl pH 8 5 fourni dans le kit ou le tampon TE voir la section 4 10 dans la seringue Placez la pointe du NucleoBond Finalizer au dessus d un nouveau tube collecteur et luez l ADN plasmidique pr cautionneusement en ins rant le piston 500 1000 ul 500 1000 pl Enlevez le NucleoBond Finalizer de la seringue retirez le piston et rattachez le NucleoBond Finalizer la sortie de la seringue l Transf rez le premier luat dans la seringue et luez dans le m me tube e collecteur une seconde fois Charger compl tement Charger compl tement le le premier luat premier luat 54 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi Midi NucleoBond Maxi NucleoBond Finalizer Finalizer Large D termination du rendement Determinez le rendement d ADN plasmidique par spectrophotom trie UV et confirmez l int grit du plasmide par lectrophor se sur gel d agarose voir section 4 11 MACHEREY NAGEL 03 2006 Rev 01 55 Plasmid DNA Purification 9 Appendix 9 1 Troubleshooting If you experience problems with reduced yield or purity it is recommended to check which purification step of the procedure is causing the problem First the bacterial culture has to be checked f
13. 4 6 44 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 7 6 Low copy plasmid purification Midi Maxi French Pr paration d une pr culture Inoculez 3 5 ml d un milieu de pr culture LB avec une colonie piqu e sur une plaque d Agar fraichement stri e Assurez vous que la plaque et le milieu de culture contiennent le bon antibiotique afin d tre s r d obtenir le plasmide pour d autres informations voir sections 4 3 Agitez a 37 C a 300 rpm pendant 8 h e Pr paration d une culture overnight grand volume Inoculez une culture overnight en diluant la pr culture au 1 1000ieme dans un volume donn de milieu LB contenant l antibiotique s lectif appropri Si la culture pr sente une faible croissance ou si le plasmide est connu pour tre faiblement repr sent consultez la section 4 5 du protocole pour l utilisation de plus grands volumes de culture Faites pousser la culture toute la nuit 37 C 300 rpm pendant 12 16 h Remarque Afin d utiliser au maximum la capacit de fixation des colonnes NucleoBond Xtra il est n cessaire de charger une quantit suffisante d ADN plasmidique Pour le protocole low copy standard le volume de culture recommand est doubl par rapport au protocole high copy Cependant tant donn que le contenu plasmidique des cellules peut tre 10 100 fois inf rieur le volume de culture peut tre insuffisant Si vous avez besoin de grand
14. 6 Rev 01 17 Plasmid DNA Purification 4 11 Determination of DNA yield and quality The yield of a plasmid preparation should be estimated prior to and after the isopro panol precipitation in order to calculate the recovery after precipitation and to find the best volume to dissolve the pellet in Just use either NucleoBond Xtra elution buffer ELU or the respective low salt buffer as a blank in your photometric measurement The nucleic acid concentration of the sample can be calculated from its UV absor bance at 260 nm where an absorbance of 1 1 cm path length is equivalent to 50 ug DNA ml Note that the absolute measured absorbance should lay between 0 1 and 0 7 to be in the linear part of Lambert Beer s law Dilute your sample in the respec tive buffer if necessary The plasmid purity can be checked by UV spectroscopy as well A ratio of Asgoiog0 between 1 80 1 90 and A5 around 2 0 indicates pure plasmid DNA An A gorso ra tio above 2 0 is a sign for too much RNA in your preparation an A goso ratio below 1 8 indicates protein contamination Plasmid quality can be checked by running the precipitated samples on a 1 aga rose gel This will give information on conformation and structural integrity of isolated plasmid DNA i e it shows whether the sample is predominantly in the favorable su per coiled ccc usually the fastest band an open circle oc or even linear form see section 9 1 Figure 3 4 12 Convenient stopping p
15. Bond Finalizer de la seringue retirez le piston et rattachez le NucleoBond Finalizer la sortie de la seringue Versez 2 ml d thanol 70 non fourni dans les kits dans la seringue ins rez le piston et pressez l thanol doucement travers le NucleoBond Finalizer Eliminez l ethanol MACHEREY NAGEL 03 2006 Rev 01 53 NucleoBond Xtra Midi Maxi Midi NucleoBond Maxi NucleoBond Finalizer Finalizer Large 4 S chage de la membrane Enlevez le NucleoBond Finalizer de la seringue retirez le piston et rattachez le NucleoBond Finalizer Faites passer de l air travers le NucleoBond Finalizer avec une force appropri e tout en touchant un tissus ou papier propre avec la pointe du NucleoBond Finalizer pour absorber l thanol R p tez cette tape au moins deux fois jusqu ce que plus aucune goutte d ethanol ne sorte du NucleoBond Finalizer Remarque une nouvelle seringue s che peut tre utilis e pour acc l rer la proc dure non fournie Option vous pouvez incuber NucleoBond Finalizer pendant 10 minutes 80 C pour minimiser la contamination thanolique Cependant le taux de r cup ration final peut tre r duit en raison d un s chage excessif de l ADN 5 Elution de l ADN Enlevez le NucleoBond Finalizer de la seringue retirez le piston d une seringue 1 ml et fixez le NucleoBond Finalizer la sortie Remarque voir la
16. Bond Finalizer f r 10 Minuten bei 80 C Zu intensives Trocknen der DNA kann allerdings zu einer reduzierten Wiederfindung f hren 5 Elution der DNA Entfernen Sie den NucleoBond Finalizer von der 30 ml Spritze ziehen Sie den Kolben einer 1 ml Spritze heraus und befestigen den NucleoBond Fi nalizer am Auslass der Spritze Hinweis Zur Wahl des geeigneten Elutionspuffer Volumens siehe Kapitel 4 9 Abbil dung 3 Midi oder 4 Maxi Pipettieren Sie redissolving buffer TRIS 5 mM Tris HCl pH 8 5 im Liefer umfang enthalten oder TE Puffer in die Spritze siehe auch Kapitel 4 10 Platzieren Sie den Auslass des NucleoBond Finalizers ber einem frischen Auffanggef und eluieren Sie die Plasmid DNA langsam 500 1000 ui 500 1000 ul Entfernen Sie den NucleoBond Finalizer von der Spritze ziehen Sie den Kolben heraus und befestigen den NucleoBond Finalizer wieder am Auslass der Spritze berf hren Sie das erste Eluat zur ck in die Spritze und eluieren ein e zweites mal in das selbe AuffanggefaB erstes Eluat erstes Eluat vollst ndig laden vollst ndig laden MACHEREY NAGEL 03 2006 Rev 01 51 NucleoBond Xtra Midi Maxi Midi NucleoBond Finalizer Maxi NucleoBond Finalizer Large 6 Bestimmung der Plasmid Ausbeute Bestimmen Sie photometrisch die Plasmidausbeute und berpr fen Sie die Plasmidintegrit t mittels Agarosegel Elektrophorese siehe K
17. H RE SPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect inciden tal compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MA CHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty ex pressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published cata logues and product literature are MACHEREY NAGEL s sole representations con cerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are author ized they should not be relied upon by the customer and are not a part of the con tract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for gen eral scientific information Applications mentioned in MACHEREY NAGEL literature are prov
18. NA If you are not sure about the plasmid copy number and growth behavior of your host strain increase the culture volume and decide in step 3 how much cells to use for the preparation Harvest bacterial cells Measure the cell culture ODgo and determine the recommended culture volume V ml 400 OD V ml 1200 OD Pellet the cells by centrifugation at 4 500 6 000 x g for 10 min at 4 C and dis card the supernatant completely Note It is of course possible to use larger culture volumes e g if the plasmid does not behave like a typical high copy vector see section 4 4 for more information In this case increase RES LYS and NEU buffer volumes proportionally in steps 4 5 and 7 If the cul ture volume is more than double the recommended culture volume it is advantageous to use a centrifuge for the lysate clarification in step 8 rather than the NucleoBond Xtra column filters MACHEREY NAGEL 03 2006 Rev 01 21 NucleoBond Xtra Midi Maxi 4 Resuspension Resuspend the cell pellet completely in resuspension buffer RES RNase A by pipetting the cells up and down For an efficient cell lysis it is important that no clumps remain in the suspension Note Increase RES buffer volume proportionally if more than the recommended cell mass is used see section 4 6 for information on optimal cell lysis and section 4 7 re garding difficult to lyse strains 9
19. S might precipitate at temperatures below 20 C If precipitation is observed incubate the bottle for several minutes at about 30 40 C and mix well until the precipitate is redissolved 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 03 2006 Rev 01 19 Plasmid DNA Purification 6 Safety instructions risk and safety phrases The following components of the NucleoBond Xtra kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section Component Hazard Hazard Risk Safety Contents Symbol Phrases Phrases RNase A RNase A x x May cause sensitization by R 42 43 S 22 24 lyophilized inhalation and skin contact LYS sodium x A Irritating to eyes and skin R 36 38 S 26 37 39 hydroxide lt 2 S 45 Risk Phrases R 36 38 Irritating to eyes and skin R 42 43 May cause sensitization by inhalation and skin contact Safety Phrases S 22 Do not breathe dust S 24 Avoid contact with the skin S 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S 37 39 Wear suitable gloves and eye face protection S 45 In case of accident or if you feel unwell seek medical advice immediately show the label where possible Label not necessary if quantity below 125 g or m
20. Sie ein gr eres Volumen Kapitel 4 4 falls die Kultur bekannterma en langsam oder schlecht w chst Inkubieren Sie auf einem Sch ttler bei 37 C und 300 rpm f r 12 16 h Hinweis Um die hohe Bindekapazit t der NucleoBond Xtra S ulen voll ausnutzen zu k nnen ist es wichtig ausreichend Plasmid DNA zu laden F r die Standard low copy Prozedur werden gegen ber dem high copy Protokoll doppelte Kulturvolumina einge setzt Trotzdem kann dies u U bei einem Plasmidgehalt der 10 100 mal geringer ist unzureichend sein Falls gro e Mengen an low copy Plasmiden ben tigt werden sollte das Kulturvolumen nochmals um einen Faktor 3 5 erh ht werden f r weitere Informatio nen siehe Kapitel 4 5 und in Schritt 3 entschieden werden wie viele Zellen f r die Pr paration eingesetzt werden MACHEREY NAGEL 03 2006 Rev 01 41 NucleoBond Xtra Midi Maxi 3 Ernte der Bakterienzellen Messen Sie die ODgo der Bakterienkultur und and bestimmen das empfohlene Kulturvolumen gem folgender Formel Pelletieren Sie die Zellen durch Zentrifugation bei 4 500 6 000 x g f r 10 min bei 4 C und entfernen Sie den berstand quantitativ Hinweis Es k nnen auch gr ere Kulturvolumina verwendet werden z B falls gro e Mengen low copy Plasmid ben tigt werden f r weitere Informationen siehe auch Kapitel 4 5 In diesem Fall erh hen Sie die Volumina der Puffer RES LYS und NEU proportio nal in den Schrit
21. Spritze F llen Sie das Pr zipitationsgemisch in die Spritze setzen Sie den Kolben ein und dr cken Sie die Suspension langsam mit konstantem Druck durch den NucleoBond Finalizer Verwerfen Sie den Durchfluss Waschen des Pr zipitats Entfernen Sie den NucleoBond Finalizer von der Spritze ziehen Sie den Kolben heraus und befestigen Sie den NucleoBond Finalizer wieder am Auslass der Spritze F llen Sie 2 ml 70 iges Ethanol nicht im Lieferumfang enthalten in die Spritze setzen Sie den Kolben ein und dr cken Sie das Ethanol langsam durch den NucleoBond Finalizer Verwerfen Sie das Ethanol 50 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi Midi NucleoBond Maxi NucleoBond Finalizer Finalizer Large 4 Trocknen der Filtermembran Entfernen Sie den NucleoBond Finalizer von der Spritze ziehen Sie den Kolben heraus und befestigen Sie den NucleoBond Finalizer wieder am Auslass der Spritze Dr cken Sie so kr ftig wie m glich Luft durch den Nuc leoBond Finalizer und nehmen das an der Spitze austretende Ethanol mit einem Tuch auf Wiederholen Sie diesen Schritt mindestens zweimal bis kein Ethanol mehr aus dem NucleoBond Finalizer austritt Hinweis Zur Beschleunigung der Prozedur kann eine neue trockene Spritze ver wendetet werden nicht im Lieferumfang enthalten Optional Um die Ethanol Verschleppung ins Eluat zu minimieren inkubieren Sie den Nucleo
22. The lysate should turn from a slimy viscous consistency to a low viscosity homogeneous suspension of an off white floccu late Immediately proceed with step 8 of the high copy plasmid purification protocol section 7 1 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 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 7 5 Low copy plasmid purification Midi Maxi German Die in den NucleoBond Xtra Kits enthaltenen Lysepuffer Volumina sind ausreichend f r die Isolierung von high copy Plasmiden Fur die Isolierung von low copy Plasmi den ist zusatzlicher Puffer notwendig Bestellinformation siehe Kapitel 9 2 Ansetzen einer Vorkultur Beimpfen Sie 3 5 ml LB Medium mit einer einzelnen Kolonie einer frisch ausge strichenen Agarplatte Stellen Sie sicher dass sowohl die Platte als auch das Fl ssigmedium das n tige Antibiotikum enth lt da bei fehlendem Selektionsdruck die Bakterien ihr Plasmid bei der Zellteilung verlieren k nnen f r weitere Infor mationen siehe Kapitel 4 3 Sch tteln Sie die Vorkultur bei 37 C und 300 rpm f r 8 h 6 Ansetzen einer bernachtkultur Beimpfen Sie LB Medium des unten angegebenen Volumens durch Verd nnen der Vorkultur um den Faktor 1 1000 Stellen Sie sicher dass das Medium das n tige Antibiotikum enth lt Wahlen
23. allung von Protein und genomischer DNA zu gew hrleisten Das schleimige viskose Lysat sollte nach Zugabe von buffer NEU d nnfl ssig werden und eine homogene Suspensi on mit flockigem weiBem Prazipitat ausbilden Fahren Sie sofort mit Schritt 8 fort Eine Inkubation des Lysates ist nicht not wendig Hinweis Hinweis Erh hen Sie das Volumen des Puffers NEU proportional falls mehr als die empfohlene Zellmasse eingesetzt wird f r Informationen zur optimalen Zelllyse siehe Kapitel 4 6 28 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 8 Kl rung des Lysates und Beladung der S ule Um eine gleichm ige Suspension des Pr zipitates zu erzielen und somit ein Verstopfen des NucleoBond Xtra Filters zu vermeiden invertieren Sie das Ge e f erneut 3 malig unmittelbar bevor Sie das Lysat auf den quilibrierten Filter geben Das Lysat wird gleichzeitig gekl rt und auf die NucleoBond Xtra S ule geladen F llen Sie den Filter nach falls mehr Lysat geladen werden soll als der Filter auf einmal fassen kann Lassen Sie die Fl ssigkeit vollst ndig durch die S ule laufen Alternativ Das Pr zipitat kann alternativ mittels Zentrifugation bei 5 000 x g f r mindestens 10 Min entfernt werden wenn z B mehr als das Doppelte der emp fohlenen Zellmasse verwendet wurde Sollte der berstand noch nicht v llig ge kl rt sein berf hren Sie ihn in ein neues Gef und wiederholen S
24. apitel 4 11 52 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 8 3 Concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers French Midi NucleoBond Maxi NucleoBond Finalizer Finalizer Large 1 Pr cipitation de ADN Ajoutez 0 7 volumes d isopropanol temp rature ambiante non fourni dans les kits Vortexez pr cautionneusement et laissez le m lange reposer pendant 2 minutes ex pour 5 ml d luat NucleoBond Xtra Midi ajoutez 3 5 ml d isopropanol pour 15 ml d luat NucleoBond Xtra Maxi ajoutez 10 5 ml d isopropanol Remarque le NucleoBond Finalizer peut retenir au maximum jusqu a 500 ug et le NucleoBond Finalizer Large est limit a 2000 ug d ADN plasmidique Verifier le contenu d ADN plasmidique de vos luats avant de passer l tape de pr cipitation en mesurant As voir la section 4 11 Charger plus d ADN peut entra ner une surcharge et une perte compl te de l chantillon 3 5 ml pour 10 5 ml pour 5 ml d luat 15 ml d luat 2 Chargement du pr cipit Enlevez le piston de la seringue 30 ml et fixez un NucleoBond Finalizer en sortie Versez le m lange de pr cipitation dans la seringue ins rez le piston et pressez le m lange afin de le faire passer doucement travers le NucleoBond Finalizer en utilisant une force constante Eliminez le filtrat 3 Lavage du pr cipit Enlevez le Nucleo
25. bes etc is clean and nuclease free Do not lyse the sample with buffer LYS for more than 5 min DNA is irreversibly denatured A denatured plasmid band runs faster on the gel than the super coiled conformation Do not lyse the sample after addition of LYS for more than 5 minutes No or low plasmid DNA yield after NucleoBond Finalizer pre cipitation Loss of eluate too high due to dead volume e Especially when you aim for high concentration you need to elute in small volumes But naturally you will lose parts of your eluate in the syringe and on the NucleoBond Finalizer To minimize these losses in the second elution step try to transfer even the last droplet from the syringe to the NucleoBond Final izer e g by tapping the NucleoBond Finalizer and syringe onto the bench top Then fill the syringe with air and press forcefully the last droplets out of the NucleoBond Finalizer Repeat this step several times You might have to practice this procedure several times to achieve optimal results An acceptable dead volume is smaller than 30 ul with NucleoBond Finalizer and 75 ul with NucleoBond Finalizer Large 62 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification Problem Possible cause and suggestions Low DNA concentration after NucleoBond Finalizer pre cipitation continued Elution volume too small e Since there are dead volumed of about 30 ul NucleoBond Fi na
26. by a sodium hydrox ide SDS treatment with buffer LYS Proteins as well as chromosomal and plasmid DNA are denatured under these conditions RNA is degraded by DNase free RNase A Neutralization buffer NEU containing potassium acetate is then added to the lysate causing SDS to precipitate as KDS potassium dodecyl sulfate and pulling down proteins chromosomal DNA and other cellular debris The potassium acetate buffer also neutralizes the lysate Plasmid DNA can revert to its native super coiled structure and remains in solution The NucleoBond Xtra buffer volumes standard protocol are adjusted to ensure optimal lysis for culture volumes appropriate for high copy plasmids according to Table 2 Using too much cell material leads to inefficient cell lysis and precipitation and might reduce your plasmid yield and purity Therefore lysis buffer volumes should be increased when applying larger culture volumes in case of e g low copy vector purification section 4 5 Table 3 By rule of thumb calculate the necessary lysis buffer volumes for RES LYS and NEU as follows Vol ml Culture Volume ml x OD 50 For example if 200 ml of a low copy bacterial culture OD 4 is to be lysed the appropriate volumes of lysis buffers RES LYS and NEU are 16 ml each If more ly sis buffer is needed than is provided with the kit an additional buffer set including buffers RES LYS NEU and RNase A can be ordered separately Please refer
27. cleoBond Xtra eluates with the NucleoBond Finalizers MACHEREY NAGEL 03 2006 Rev 01 oo nN Oo 11 12 13 14 14 15 16 16 18 18 19 20 21 21 26 32 38 41 45 48 NEW NEW Plasmid DNA Purification 8 1 Concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers English 8 2 Concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers German 8 3 Concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers French 9 Appendix 9 1 Troubleshooting 9 2 Ordering information 9 3 Product use restriction warranty 4 MACHEREY NAGEL 03 2006 Rev 01 48 50 53 56 56 64 64 Plasmid DNA Purification 1 Kit contents NucleoBond Xtra NucleoBond Xtra Midi Midi Plus 10 preps 50 preps 100 preps 10 preps 50 preps Cat No 740410 10 740410 50 740410 100 740412 10 740412 50 Buffer RES 100 ml 500 ml 1000 ml 100 ml 500 ml Buffer LYS 4 x 25 ml 500 ml 1000 ml 4 x 25 ml 500 ml Buffer NEU 100 ml 500 m 1000 ml 100 ml 500 ml Buffer EQU 200 ml 2 x 500 ml 2x 1000 ml 200 ml 2 x 500 ml Buffer WASH 100 ml 500 ml 1000 ml 100 ml 500 ml Buffer ELU 60 ml 300 ml 600 ml 60 ml 300 ml RNase A lyophilized 6 mg 30 mg 2 x 30 mg 6 mg 30 mg NucleoBond Xtra 10 50 100 10 50 Midi columns NucleoBond Xtra Midi column filters 10 50 100 10 50 NucleoBond U g 10 50 Finalizer 30 ml Syringes 10 50 1 ml Syringes 10 50 Buffe
28. command e 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 a lyser Lyse cellulaire Avant d utiliser le tampon LYS v rifiez que le SDS n a pas pr cipit Si un precipite blanc est visible chauffer le tampon quelques minutes a 30 40 C e jusqu ce que le pr cipit soit compl tement dissout Ramenez le tampon a temp rature ambiante 20 25 C Ajoutez le tampon de lyse lysis buffer LYS la suspension M langez avec pr caution en inversant le tube 5 fois Ne pas utiliser de vortex sinon l ADN chromosomique se fractionnerait se detacherait des d bris cellulaires et contaminerait alors la suspension Incubez le m lange temp rature ambiante 20 25 C pendant 5 min Remarque augmentez proportionnellement le volume de tampon LYS si vous avez utilis une masse cellulaire sup rieure celle recommand e voir la section 4 6 pour plus d informations sur la lyse optimale des cellules 46 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 6 Equilibration Equilibrez la colonne NucleoBond Xtra avec le filtre int gr avec le tampon d quilibration equilibration buffer EQU D posez le tampon sur la collerette du filtre comme le montre le sch ma Laissez la colonne se vider par gravit et assurez vous que le filtre est enti rement mouill Ne pas laisser la co
29. conditions shaking temperature host strain or type of plasmid insert etc the final amount of cells in a bacterial culture can vary over a wide range E g overnight cultures in flasks usually reach an OD of 3 6 under vigorous shaking while fermentation cul tures reach an OD of 10 and more By rule of thumb 1 liter of E coli culture grown in LB medium yields a pellet of about 5 10 g wet weight The expected DNA yield for a high copy plasmid is 1 mg per gram cell wet weight It is therefore important to adjust the cell mass rather than the culture volume for the best plasmid purification results But since the cell mass or cell wet weight is tedious to determine it was replaced in this manual by the mathematical product of optical density at 600 nm OD and culture volume V two variables that are much easier to measure ODV OD x Vol mi Table 2 Recommended culture volumes for high copy plasmids Pellet Recommended culture volume for NucleoBond wel Rec Xtra Kit weight ODV OD oc OD soc OD 500 OD 500 OD Midi 0 759 400 200 ml 100 ml 66 ml 50 ml 40 ml Maxi 2 259 1200 600 ml 300 ml 200 ml 150 ml 120 ml Table 2 shows recommended ODVs and the corresponding pairs of ODeo and cul ture volume that can be easily handled using the standard kit protocol Iysis buffer volumes 12 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification 4 5 Culture volume for l
30. d calculate the recovery after precipitation 24 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 13 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Vor tex well and let the mixture sit for 2 minutes Centrifuge at 5 000 x 9 for 215 min at lt room temperature preferably at 15 000 x g for 30 min at 4 C Carefully discard the supernatant 14 Wash and dry DNA pellet Add room temperature 70 ethanol to the pellet and centrifuge at 5 000 x g preferably 15 000 x g for 5 min at room temperature 20 25 C 2 ml Carefully remove ethanol completely from the tube with a pipette tip Allow the pellet to dry at room temperature 20 25 C Note Plasmid DNA might be harder to dissolve when over dried 15 Reconstitute DNA Dissolve the DNA pellet in an appropriate volume of buffer TE or sterile deionized H O Depending on the type of centrifugation tube dissolve under gentle pipetting up and down or constant spinning in a sufficient amount of buffer for 10 60 min 3D shaker Determine plasmid yield by UV spectrophotometry Confirm plasmid integrity by agarose gel electrophoresis see section 4 11 MACHEREY NAGEL 03 2006 Rev 01 25 NucleoBond Xtra Midi Maxi 7 2 High copy plasmid purification Midi Maxi German Ansetzen einer Vorkultur Beimpfen Sie 3 5 ml LB Me
31. dium mit einer einzelnen Kolonie einer frisch ausge strichenen Agarplatte Stellen Sie sicher dass sowohl die Platte als auch das Fl ssigmedium das n tige Antibiotikum enth lt da bei fehlendem Selektionsdruck die Bakterien ihr Plasmid bei der Zellteilung verlieren k nnen f r weitere Informa tionen siehe Kapitel 4 3 Sch tteln Sie die Vorkultur bei 37 C und 300 rpm f r 8 h OS 22 Ansetzen einer bernachtkultur Beimpfen Sie LB Medium des unten angegebenen Volumens durch Verd nnen der Vorkultur um den Faktor 1 1000 Stellen Sie sicher dass das Medium das n tige Antibiotikum enth lt W hlen Sie ein gr eres Volumen Kapitel 4 4 falls die Kultur bekannterma en langsam oder schlecht w chst oder das Plasmid sich nicht wie ein high copy Plasmid verh lt Inkubieren Sie auf einem Sch ttler bei 37 C und 300 rpm f r 12 16 h Hinweis Um die hohe Bindekapazit t der NucleoBond Xtra S ulen voll ausnutzen zu k nnen ist es wichtig ausreichend Plasmid DNA zu laden Sollten Sie bzgl Kopienzahl des Plasmids und Wachstumsverhaltens des Bakterienstamms unsicher sein erh hen Sie das Kulturvolumen und entscheiden in Schritt 3 wie viele Zellen f r die Pr paration eingesetzt werden 26 Ernte der Bakterienzellen Messen Sie die ODs der Bakterienkultur und bestimmen Sie das empfohlene Kulturvolumen gem folgender Formel V ml 400 OD V ml 1200 OD Pelletieren Sie die Zellen du
32. e 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 11 MACHEREY NAGEL 03 2006 Rev 01 49 NucleoBond Xtra Midi Maxi 8 2 Concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers German Midi NucleoBond Maxi NucleoBond Finalizer Finalizer Large Pr zipitation Pr zipitieren Sie die eluierte Plasmid DNA durch Zugabe von 0 7 Volumen Isopropanol nicht im Lieferumfang enthalten wobei der Alkohol Raum temperatur haben sollte Mischen Sie sofort gr ndlich durch Vortexen und lassen Sie die Mischung f r 2 Minuten stehen Geben Sie z B 3 5 ml Isopropanol zu 5 ml NucleoBond Xtra Midi Eluat oder 10 5 ml Isopropanol zu 15 ml NucleoBond Xtra Maxi Eluat Hinweis Die Bindekapazit t des NucleoBond Finalizers betr gt 500 ug Plasmid DNA die des NucleoBond Finalizer Large betr gt 2000 ug berpr fen Sie den Plasmidgehalt des Eluates vor der Pr zipitation durch Bestimmung des Azs siehe Kapitel 4 11 Die Beladung mit gr eren DNA Mengen kann zum Verstopfen des NucleoBond Finalizers und somit zum kompletten Verlust Ihrer Probe f hren Laden des Pr zipitats Ziehen Sie den Kolben einer 30 ml Spritze vollst ndig heraus und befestigen Sie den NucleoBond Finalizer am Auslass der
33. e has been analyzed on a 1 agarose gel Equal amounts of plasmid DNA before lane 1 and after lane 4 purification using NucleoBond Xtra Midi are shown with a recovery of gt 90 M 12345 Der w M Marker Hindill cleared lysate ccc linear and oc structure of the plasmid degraded RNA Il lysate flow through no plasmid DNA but degraded RNA Ill wash flow through no plasmid DNA or residual RNA IV eluate pure plasmid DNA EcoRI restriction linearized form of plasmid MACHEREY NAGEL 03 2006 Rev 01 57 Plasmid DNA Purification Problem Possible cause and suggestions No or low plasmid DNA yield Plasmid did not propagate Check plasmid content in the cleared lysate by precipitation of an aliquot Use colonies from fresh plates for inoculation and add selective antibiotic to plates and media e Estimate plasmid content prior to large purifications by a quick NucleoSpin Plasmid or NucleoSpin Plasmid QuickPure prepa ration Alkaline lysis was inefficient Too much cell mass was used Refer to section 4 4 4 6 regard ing recommended culture volumes and lysis buffer volumes Check buffer LYS for SDS precipitation before use especially after storage below 20 C If necessary incubate the bottle for several minutes at 30 40 C and mix well until SDS is re dissolved SDS or other precipitates are present in the sample Load the crude lysate onto the NucleoBond Xtra
34. e plasmid DNA that should be recovered after precipitation Plasmid DNA might be smeared over the wall of the tube e Dissolve DNA with an appropriate volume of TE buffer by rolling the tube for at least 30 min Nucleic acid did not precipitate Check type and volumes of precipitating solvent Make sure to use at least 0 7 volumes of isopropanol e Centrifuge for longer periods of time at higher speed Nucleic acid pellet does not resuspend in buffer Pellet was over dried Try to dissolve at higher temperatures for a longer period of time e g 2 h at 37 C or overnight at RT preferably under constant spinning 8D shaker Co precipitation of salt or residual alcohol e Wash the pellet again with 70 ethanol or increase the recon stitution buffer volume MACHEREY NAGEL 03 2006 Rev 01 61 Plasmid DNA Purification Problem Possible cause and suggestions Purified plas mid does not perform well in subsequent reactions Plasmid DNA is contaminated with chromosomal DNA or RNA e Refer to the detailed trouble shooting above Plasmid DNA is contaminated with residual alcohol Plasmid DNA was not dried completely before redissolving Pre cipitate DNA again by adding 1 10 volume of 3 M NaAc pH 5 0 and 0 7 volumes of isopropanol Proceed with the precipitation protocol in this manual und dry DNA Pellet completely DNA is degraded Make sure that your entire equipment pipettes centrifuge tu
35. e strains Cell lysis Check lysis buffer LYS for precipitated SDS prior to use If a white precipitate is visible warm the buffer for several minutes at 30 40 C until precipitate is dis solved completely Cool buffer down to room temperature 20 25 C Add Iysis buffer LYS to the suspension Mix gently by inverting the tube 5 times Do not vortex as this will shear and release contaminating chromosomal DNA from cellular debris into the suspension Incubate the mixture at room temperature 20 25 C for 5 min Note Increase LYS buffer volume proportionally if more than the recommended cell mass is used see section 4 6 for information on optimal cell Iysis Equilibration Equilibrate a NucleoBond Xtra column together with the inserted column filter with equilibration buffer EQU 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 ea ani MACHEREY NAGEL 03 2006 Rev 01 39 NucleoBond Xtra Midi Maxi 7 D 40 Neutralization Add neutralization buffer NEU to the suspension and immediately mix the lysate gently by inverting the tube 10 15 times Do not vortex The flask or tube used for this step should not be filled more than two thirds to allow homogeneous mixing Make sure to neutralize completely to precipitate all the protein and chromosomal DNA
36. ension Incubez le m lange temp rature ambiante 20 25 C pendant 5 min Remarque Augmentez proportionnellement le volume du Tampon LYS si vous avez utilis une masse cellulaire sup rieure celle recommand e voir section 4 6 pour plus d informations sur la lyse optimale des cellules MACHEREY NAGEL 03 2006 Rev 01 33 NucleoBond Xtra Midi Maxi 6 Equilibration Equilibrez la colonne NucleoBond Xtra avec le filtre int gr avec le tampon d quilibration equilibration buffer EQU D posez le tampon sur la collerette du filtre comme le montre le sch ma Laissez la colonne se vider par gravit et assurez vous que le filtre est enti rement mouill Ne pas laisser la colonne s ass cher 7 Neutralisation m Ajoutez le tampon de neutralisation neutralization buffer NEU la suspension et m langez imm diatement avec pr caution le lysat en inversant le tube 10 15 fois Ne pas utiliser de vortex Le flacon ou le tube utilis pour cette tape ne doit pas tre rempli au plus des deux tiers afin de permettre un m lange homog ne Veillez neutraliser compl tement pour pr cipiter toutes les prot ines et l ADN chromosomique Le lysat doit passer d une forme gluante et visqueuse une forme moins visqueuse une suspension homog ne et un floculat blanc Proc dez imm diatement l tape 8 Une incubation du lysat n est pas n cessaire Remarque Aug
37. ent le filtre s il est inutilise Laissez la colonne se vider par gravite Remarque Vous pouvez garder tous les filtrats pour les analyser voir section 9 1 o lt nb Lavage du filtre et de la colonne equilibration buffer EQU Lavez le filtre NucleoBond Xtra et la colonne NucleoBond Xtra avec le tampon equilibration buffer EQU Deposez le tampon sur la collerette du filtre et assurez vous que le lysat est bien lav Omettre cette tape ou d poser simplement le tampon dans le filtre r duit le rendement d ADN plasmidique 10 Eliminer le filtre Ly 4 Retirez le filtre NucleoBond Xtra ou liminez le en retournant la colonne MACHEREY NAGEL 03 2006 Rev 01 35 NucleoBond Xtra Midi Maxi 11 Lavage de la colonne washing buffer WASH Lavez la colonne NucleoBond Xtra avec le tampon de lavage washing buffer WASH Il est important d liminer le filtre avant d appliquer le tampon de lavage En effet ceci pourrait avoir un mauvais impact sur la puret de l ADN 12 Elution Eluez l ADN plasmidique avec le tampon d lution elution buffer ELU Passez l etape 13 pour le protocole de centrifugation apr s la pr cipitation a l isopropanol ou continuez avec la section 8 pour la concentration de ADN plasmidique et son d salage au moyen du syst me NucleoBond Finalizer NucleoBond Xtra Midi Plus ou NucleoBond Finalizer Large N
38. eoBond Xtra column Compared to lysate clearing by centrifugation or syringe filters the NucleoBond Xtra column filter furthermore avoids shearing of large DNA constructs such as PACs or BACs by the gentle depth filter effect filtration occurs on the surface of the filter as well as inside the filter matrix Its special material and design lead to very rapid passage of the lysate through the filter and even very large lysate volumes can be applied without the risk of clogging This is especially important for e g low copy plasmid purification However if more than the recommended cell mass see section 4 4 Table 2 section 4 5 Table 3 was lysed it might be advantageous to use a cen trifuge for lysate clarification rather than the provided column filters due to their lim ited precipitate capacity MACHEREY NAGEL 03 2006 Rev 01 15 Plasmid DNA Purification 4 9 Washing of the column The high salt concentration of the lysate prevents proteins and RNA from binding to the NucleoBond Xtra column see section 4 2 Figure 2 However to remove all traces of contaminants and to purge the dead volume of the NucleoBond Xtra col umn filters it is important to wash the column and the filter in two subsequent wash ing steps First apply buffer EQU to the funnel rim of the filter to wash all residual lysate out of the filter onto the column Do not just pour the buffer inside the filter Then pull out and discard the column filter
39. erforming a Midi prep and in 12 ml for a Maxi prep respectively Follow the handling instructions exactly and note the given hints for protocol alterations 8 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification 4 NucleoBond Xtra plasmid purification system 4 1 Basic principle The bacterial cells are lysed by an optimized set of newly formulated buffers based on the NaOH SDS lysis method of Birnboim and Doly After equilibration of the NucleoBond Xtra column together with the corresponding NucleoBond Xtra column filter the entire lysate is loaded by gravity flow and si multaneously cleared by the newly designed column filter Plasmid DNA is bound to the improved NucleoBond Xtra silica resin After an efficient washing step the plasmid DNA is eluted precipitated and easily dissolved in any suitable buffer e g low salt buffer or water for further use 4 2 NucleoBond Xtra anion exchange columns NucleoBond Xtra is a silica based anion exchange resin developed by MACHEREY NAGEL and covered under European Patent EP 0496822 It is devel oped for routine separation of different classes of nucleic acids like oligonucleotides RNA and plasmids NucleoBond Xtra silica resin consists of hydrophilic macroporous silica beads functionalized with MAE methyl amino ethanol The dense coating of this functional group provides a high overall positive charge density under acidic pH conditions that permits the nega
40. es quantit s de plasmides low copy augmentez nouveau le volume de culture d un facteur 3 5 voir la section 4 5 pour plus d informations et d cidez l tape 3 de la quantit de cellules a utiliser pour la pr paration R cup ration des cellules bact riennes Mesurez la DO de la culture cellulaire et d terminez le volume de culture recommand Culottez les cellules par centrifugation 4 500 6 000 x g pendant 10 min 4 C et liminez le surnageant compl tement Remarque il est possible bien s r d utiliser de plus grands volumes de culture par ex si un grande quantit de plasmides low copy doit tre r cup r e voir la partie 4 5 pour plus d informations Dans ce cas augmentez les volumes des tampons RES LYS et NEU proportionnellement aux tapes 4 5 et 7 et utilisez la centrifugation pour la clarification du lysat plut t que les colonnes filtres NucleoBond Xtra column filters MACHEREY NAGEL 03 2006 Rev 01 45 NucleoBond Xtra Midi Maxi 4 5 Resuspension Rependre compl tement le culot cellulaire dans le tampon de resuspension re suspension buffer RES RNase A en pipettant par aspiration refoulement ou en vortexant Important pour une lyse efficace des cellules il ne doit plus subsister d agr gats en suspension Remarque augmentez proportionnellement le volume de tampon RES si vous avez utilis une masse cellulaire sup rieure a celle re
41. fy lysate prepared from a culture volume larger than recommended for any given column size with stan dard lysis buffer volumes Incomplete lysis not only blocks the column but can also significantly reduce yields Refer to section 4 4 and 4 5 for recommended culture volumes and section 4 6 for larger culture volumes and adjusted lysis buffer volumes NucleoBond Xtra column is Make sure to mix well after neutralization to completely precipi blocked or tate SDS and chromosomal DNA very slow Lysate was not cleared completely Use NucleoBond Xtra column filter or centrifuge at higher speed or for a longer period of time Precipitates occur during storage Clear lysate again before loading the column Lysis treatment was too harsh Make sure not to lyse in buffer LYS for more than 5 min Lysate was mixed too vigorously or vortexed after lysis Genomic DNA Invert tube for only 5 times Do not vortex after addition of LYS or RNA con tamination of Use larger tubes or reduce culture volumes for easier mixing plasmid DNA RNase digestion was inefficient RNase was not added to buffer RES or stored improperly Add new RNase to buffer RES See section 9 2 for ordering informa tion MACHEREY NAGEL 03 2006 Rev 01 59 Plasmid DNA Purification Problem Possible cause and suggestions Low purity A230 A280 lt 1 8 NucleoBond Xtra column filter was not removed before second washing step e Protein content too
42. he plasmid DNA Whereas strains like DH5a or XL1 Blue usually produce high quality super coiled plasmid DNA other strains like e g HB101 with high levels of endonuclease activity might yield lower quality plasmid giving poor results in downstream applications like enzy matic restriction or sequencing Maniatis T Fritsch EF Sambrook J Molecular cloning A laboratory manual Cold Spring Harbor Cold Spring New York 1982 MACHEREY NAGEL 03 2006 Rev 01 11 Plasmid DNA Purification The type of plasmid especially the size and the origin of replication ori has a crucial influence on DNA yield In general the larger the plasmid or the cloned insert is the lower is the expected DNA yield due to a lower copy number Even a high copy construct based on a ColE1 ori can behave like a low copy vector in case of a large or unfavorable insert In addition the ori itself influences the yield by factor 10 100 Thus plasmids based on e g pBR322 or pACYC cosmids or BACs are main tained at copy numbers lt 20 down to even only 1 whereas vectors based on e g pUC pBluescript or p EM can be present in several hundred copies per cell Therefore all the above mentioned factors should be taken into consideration if a particular DNA yield has to be achieved Culture volume and lysis procedures have to be adjusted accordingly 4 4 Culture volume for high copy plasmids Due to the influence of growth media TB CircleGrow 2xYT growth
43. ided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correct ness of any of those applications Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BIO mn net com MACHEREY NAGEL 03 2006 Rev 01 65
44. ie die Zentri fugation vorzugsweise bei h herer Geschwindigkeit oder geben Sie ihn auf den NucleoBond Xtra Filter Diese Lysatkl rung ist sehr wichtig da restliches Pr zipitat die NucleoBond Xtra S ule verstopfen k nnte Um die S ule zu beladen kann das geklarte Lysate entweder auf den aquilibrierten Filter oder nach Entfernung des unbenutzten Fil ters direkt auf die quilibrierte S ule geladen werden Lassen Sie die Fl ssigkeit vollst ndig durch die S ule laufen Hinweis An diesem Punkt der Prozedur kann ein Teil bzw der gesamte Durchfluss der S ule f r analytische Zwecke aufgehoben werden siehe Kapitel 9 1 9 Waschen des Filters und der S ule equilibration buffer EQU Waschen Sie den NucleoBond Xtra Filter und die NucleoBond Xtra S ule mit equilibration buffer EQU Geben Sie den Puffer auf den u eren Rand des S ulen filters wie in der Abbildung rechts gezeigt und stellen Sie sicher dass das im Filter verbliebene Lysat ausgewa schen wird Wird dieser Schritt weggelassen oder der Puffer direkt in den S ulenfilter statt auf den Rand gege ben kann dies zu reduzierter Plasmid ausbeute f hren MACHEREY NAGEL 03 2006 Rev 01 29 NucleoBond Xtra Midi Maxi 10 Entfernen des Filters Ir Entnehmen Sie den NucleoBond Xtra Filter oder entfernen Sie ihn durch einfaches Umdrehen der S ule Bi 11 oo zxE entfernen da sonst die Reinheit der DNA negativ
45. ifugieren Sie bei 5 000 x g vorzugsweise 15 000 x g f r 5 min bei Raumtemperatur 20 25 C 2 mi Entfernen Sie das Ethanol vollstandig mit Hilfe einer Pipette Lassen Sie das Pel let bei Raumtemperatur 20 25 C an der Luft trocknen Hinweis Zu langes Trocknen des Pellets kann das anschlie ende L sen der DNA er schweren 15 L sen der DNA L sen Sie das DNA Pellet in einem geeigneten Volumen TE Puffer oder in steri lem deionisiertem H O Je nach Art des verwendeten Zentrifugationsgef es sollte die DNA unter vorsichtigem Auf und Abpipettieren oder unter gleichm i gem Sch tteln in einem ausreichenden Volumen Puffer f r 10 60 Min 3D Sch ttler erfolgen Bestimmen Sie photometrisch die Plasmidausbeute und berpr fen Sie die Plas midintegrit t mittels Agarosegel Elektrophorese siehe Kapitel 4 11 MACHEREY NAGEL 03 2006 Rev 01 31 NucleoBond Xtra Midi Maxi 7 3 High copy plasmid purification Midi Maxi French 1 Pr paration d une pr culture Inoculez 3 5 ml d un milieu de pr culture LB avec une colonie piqu e sur une plaque d Agar fraichement stri e Assurez vous que la plaque et le milieu de culture contiennent le bon antibiotique afin d tre s r d obtenir le plasmide pour d autres informations voir section 4 3 Agitez 37 C a 300 rpm pendant 8 h 2 Pr paration d une culture overnight Inoculez une culture overnight en di
46. ity flow Note You may want to save all or part of the flow through for analysis see section 9 1 MACHEREY NAGEL 03 2006 Rev 01 23 NucleoBond Xtra Midi Maxi 9 Wash column filter and column equilibration buffer EQU Wash the NucleoBond Xtra column filter and Nu cleoBond Xtra column with equilibration buffer EQU Apply the buffer to the funnel shaped rim of the filter and make sure it is washing out the lysate which is remaining in the filter Omitting this step or just pouring the buffer directly inside the funnel may reduce plasmid yield 5 ml 10 Discard column filter Either pull out the NucleoBond Xtra column filter or discard it by H turning the column upside down A P e 11 important to remove the column filter before applying the washing buffer to avoid low purity Wash column washing buffer WASH Wash the NucleoBond Xtra column with washing buffer WASH It is g 8 ml 12 Elution Elute the plasmid DNA with elution buffer ELU Proceed with step 13 for the centrifugation protocol after isopropanol precipitation or continue with section 8 for plasmid concentration and desalination by using the NucleoBond Finalizer NucleoBond Xtra Midi Plus or NucleoBond Finalizer Large NucleoBond Xtra Maxi Plus Optional Determine plasmid yield by UV spectrophotometry in order to adjust desired concentration of DNA in step 15 an
47. izers are designed for quick concentration and desalination of plasmid and cosmid DNA eluates that are obtained by anion exchange chromatog raphy based DNA purification The sample is precipitated with isopropanol as men tioned above and loaded onto a special silica membrane by means of a syringe After an ethanolic washing step the membrane is dried by pressing air through the filter Elution of pure DNA is carried out with slightly alkaline low salt buffers like buffer TRIS 5 mM Tris HCl pH 8 5 provided with the NucleoBond Xtra Plus kits or TE buffer 10 mM Tris HCl pH7 5 1 mM EDTA For maximum yield it is recommended to perform the elution step twice The first elution step is done using fresh buffer whereas in the second elution step the eluate from the first elution is reapplied on the NucleoBond Finalizer to allow com plete solubilization of the plasmid DNA recovery highly depends on the used elution buffer volume Large volumes result in a high recovery of up to 90 but a lower DNA concentration Small elution volumes on the other hand increase the concentration but at the cost of DNA yield 16 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification Refer to Figure 3 and Figure 4 to select an elution buffer volume that meets your needs best If a small volume is chosen make sure to recover as much eluate as possible from the syringe and NucleoBond Finalizer by pressing air through the NucleoBond Finalizer se
48. l according to 67 548 EEC Art 25 1999 45 EC 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 20 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 7 NucleoBond Xtra plasmid purification The following section includes the protocols for high copy and low copy plasmid puri fication in English German and French 7 1 High copy plasmid purification Midi Maxi English Prepare a starter culture Inoculate a 3 5 ml starter culture of LB medium with a single colony picked from a freshly streaked agar plate Make sure that plate and liquid culture contain the appropriate selective antibiotic to guarantee plasmid propagation see section 4 3 for more information Shake at 37 C and 300 rpm for 8 h Prepare a large overnight culture Inoculate an overnight culture by diluting the starter culture 1 1000 into the given volumes of LB medium also containing the appropriate selective antibiotic If the culture is Known to grow poorly or the plasmid does not quite behave like a high copy plasmid please consult section 4 4 for larger culture volumes Grow the culture overnight at 37 C and 300 rpm for 12 16 h Note To utilize the entire large binding capacity of the NucleoBond Xtra columns it is important to provide enough plasmid D
49. lar biology applications including transfection in vitro transcription automated or manual sequencing cloning hybridization and PCR _ Plasmid DNA 4 DNA Single stranded DNA gt Double stranded DNA m mRNA 16S 23S rRNA _ 5S rRNA Compound class lt tRNA Proteins dyes polysaccharides metabolites trinucleotides rRNA Plasmid DNA Absorbance at 260 nm 0 0 5 1 1 5 2 Salt concentration for elution M KCI Figure 2 Elution profile of NucleoBond Xtra silica resin at pH 7 0 The more interactions a nucleic acid can form between phosphate backbone and the posi tively charged resin the later it is eluted with increasing salt concentration Large nucleic acids carry more charges than short ones double stranded DNA more than single stranded RNA 10 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification 4 3 Growth of bacterial cultures Yield and quality of plasmid DNA highly depend on the type of culture media and antibiotics the bacterial host strain the plasmid type size and copy number For standard high copy plasmids LB Luria Bertani medium is recommended The cell culture should be incubated at 37 C with constant shaking 200 250 rpm pref erably 12 16 h over night Use flasks of at least three or four times the volume of the culture volume to provide a growth medium saturated with oxygen Alternatively rich media like 2xYT Yeast Tryptone TB Terrific Broth or CircleG
50. lizer and 75 ul NucleoBond Finalizer Large reasonable elution volumes start with 100 pl NucleoBond Finalizer and 400 ul NucleoBond Finalizer Large Furthermore smaller vol umes are insufficient to wet the entire membrane and will drasti cally decrease your yield Forgot to elute a second time Repeating the elution procedure with the first eluate is crucial for optimal yields However eluting a third time shows no further improvement Fresh elution buffer was used for second elution step The second elution step is crucial for optimal yield but to achieve a high DNA concentration the eluate of the first elution step has to be used for the second elution Plasmid size Precipitation efficiency is almost independent of plasmid size but elution becomes more and more difficult for larger con structs If you face low yields with large cosmids you may try heating the NucleoBond Finalizer the syringes and elution buffer to 70 C Already no or low plasmid DNA after elution Refer to detailed trouble shooting No or low plasmid DNA yield Not enough DNA loaded Since there is a technical limitation to at least 100 ul Nucleo Bond Finalizer and 400 ul NucleoBond Finalizer Large of elution buffer due to membrane wetting and dead volume a minimal amount of DNA has to be loaded to achieve a desired concentration If possible try to pool several DNA precipitation batches since percentage of recovery and c
51. lonne s ass cher 7 Neutralisation Ajoutez le tampon de neutralisation neutralization buffer NEU la suspension et m langez imm diatement avec pr caution le lysat en inversant le tube 10 15 fois Ne pas utiliser de vortex Le flacon ou le tube utilis pour cette tape ne doit pas tre rempli au plus des deux tiers afin de permettre un m lange homog ne Veillez neutraliser compl tement pour pr cipiter toutes les prot ines et l ADN chromosomique Le lysat doit passer d une forme gluante et visqueuse une forme moins visqueuse une suspension homog ne et un floculat blanc Passez l tape 8 du protocole de purification des plasmides high copy section 7 1 Une incubation du lysat n est pas n cessaire Remarque augmentez proportionnellement le volume de tampon NEU si vous avez utilis une masse cellulaire sup rieure celle recommand e voir la section 4 6 pour plus d informations sur la lyse optimale des cellules MACHEREY NAGEL 03 2006 Rev 01 47 NucleoBond Xtra Midi Maxi 8 Concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers The following section includes the protocols for concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers in English German and French 8 1 Concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers English Midi NucleoBond Maxi NucleoBond Finalizer Finalizer Large 1 Precipitate DNA
52. luant la pr culture au 1 1000ieme dans un volume donn de milieu LB contenant l antibiotique s lectif appropri Si la culture pr sente une faible croissance ou si le plasmide ne se comporte pas comme un plasmide high copy consultez la section 4 4 du protocole pour l utilisation de plus grands volumes de culture e Faites pousser la culture toute la nuit a 37 C a 300 rpm pendant 12 16 h Remarque Afin d utiliser au maximum la capacit de fixation des colonnes NucleoBond Xtra il est n cessaire de charger une grande quantit d ADN plasmidique Si vous n tes pas s r du nombre de copies de votre plasmide ou du comportement de la culture vis a vis de la croissance bact rienne augmentez le volume de culture et d cidez l tape 3 du nombre de cellules utiliser pour la pr paration 3 R cup ration des cellules bact riennes Mesurez la DO de la culture cellulaire et d terminez le volume de culture recommand V ml 400 OD V ml 1200 OD Culottez les cellules par centrifugation a 4 500 6 000 x g pendant 10 min a 4 C et liminez compl tement le surnageant Remarque II est possible bien s r d utiliser de plus grands volumes par ex si le plasmide ne se comporte pas comme un vecteur high copy voir section 4 4 pour plus d informations Dans ces conditions il est n cessaire d augmenter proportionnellement les volumes des tampons RES LYS e
53. mentez proportionnellement le volume du Tampon NEU si vous avez utilis une masse cellulaire sup rieure celle recommand e voir section 4 6 pour des informations sur la lyse optimale des cellules 34 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 8 Clarification et chargement Assurez vous d avoir une suspension homog ne du pr cipit en inversant le tube 3 fois avant d appliquer directement le lysat sur le filtre NucleoBond Xtra pr alablement quilibr afin d viter que le filtre ne se bouche Le lysat est simultan ment clarifi et charg sur la colonne Rechargez le filtre si le volume de lyse est plus important que la capacit du filtre Laissez la colonne se vider par gravite Alternative Le pr cipit peut tre limin par centrifugation 25 000 x g pendant au moins 10 mn par ex si plus du double de la masse cellulaire recommand e a t utilis S il reste des mati res en suspension transf rez dans un nouveau tube et r p tez l tape de centrifugation de pr f rence une vitesse plus lev e ou chargez le lysat sur le filtre NucleoBond Xtra pr alablement quilibr Cette tape de clarification est extr mement importante En effet les r sidus du pr cipit peuvent boucher la colonne NucleoBond Xtra Pour charger la colonne vous pouvez appliquer le lysat clarifi sur le filtre pr alablement quilibr ou enlever pr alablem
54. oints Cell pellets can easily be stored for several months at 20 C Cleared lysates can be kept on ice or at 4 C for several days Once the column purification is started it should not be interrupted for more than an hour The columns can be left unattended for several minutes since they do not run dry but complete drying out due to evaporation should be avoided at any rate You should proceed to the elution step after which the eluate can be stored for several days at 4 C Note that the eluate should be warmed up to room temperature before precipitating the DNA to avoid co precipitation of salt 18 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification 5 Storage conditions and preparation of working solutions All kit components can be stored at room temperature 20 25 C and are stable up to two years Before you start any NucleoBond Xtra plasmid purification prepare the following e Dissolve the lyophilized RNase A by the addition of 1 ml of buffer RES Wearing gloves is recommended Pipette up and down until the RNase A is dissolved completely Transfer the RNase A solution back to the bottle con taining buffer RES and shake well Note the date of RNase A addition The fi nal concentration of RNase A is 60 ug ml buffer RES Store buffer RES with RNase A at 4 C The solution will be stable at this temperature at least up to 6 months Buffer LYS should be stored at room temperature 20 25 C since the con taining SD
55. oncentration signifi cantly increase with higher amounts of loaded DNA MACHEREY NAGEL 03 2006 Rev 01 63 Plasmid DNA Purification 9 2 Ordering information Product Cat No Pack of NucleoBond Xtra Midi 740410 10 10 preps NucleoBond Xtra Midi 740410 50 50 preps NucleoBond Xtra Midi 740410 100 100 preps NucleoBond Xtra Midi Plus 740412 10 Pe cite de NucleoBond Xtra Midi Plus 740412 50 ne NucleoBond Xtra Maxi 740414 10 10 preps NucleoBond Xtra Maxi 740414 50 50 preps NucleoBond Xtra Maxi 740414 100 100 preps NucleoBond Xtra Maxi Plus 740416 10 I LO BR NucleoBond Xtra Maxi Plus 740416 50 a LL NucleoBond Xtra combi rack 740415 1 NucleoBond Xtra buffer set 740417 1 NucleoBond Finalizer 740519 20 20 filters 2 syringe sets NucleoBond Finalizer Plus 740520 20 20 sets 20 syringe sets RNase A 740505 100 mg RNase A 740505 50 50 mg 9 3 Product use restriction warranty NucleoBond Xtra Midi Maxi kit components were developed designed and sold for research purposes only They are suitable for in vitro uses only No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking The NucleoBond Finalizer Finalizer Plus kits are suitable for use with NucleoBond Xtra Midi kits only see section 4 9 64 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification It is rather the resp
56. onsibility of the user to verify the use of the NucleoBond Xtra Midi Maxi kit for a specific application range as the performance characteristic of this kit has not been verified to a specific organism 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 Supplementary reference is made to the general business 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 against defects in products or components not manufactured by MACHEREY NAGEL or against damages resulting from such non MACHEREY NAGEL compo nents or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPE CIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY RE PRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WIT
57. or remove the filter by turning the column upside down 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 4 10 Elution and concentration of plasmid DNA Elution is carried out under high salt conditions and by a shift of pH from 6 5 to 9 0 Under these alkaline conditions the positive charge of the anion exchange resin is neutralized and plasmid DNA is released For any downstream applications it is nec essary to precipitate the DNA and to remove salt and all traces of alcohol since they disturb or inhibit enzymatic activity needed for restriction or sequencing reactions All NucleoBond Xtra eluates already contain enough salt for an isopropanol pre cipitation of DNA Therefore the precipitation is started by directly adding 0 7 volumes of isopropanol To prevent co precipitation of salt use room temperature 20 25 C isopropanol only and do not let the plasmid DNA solution drop into a vial with iso propanol but add isopropanol to the final eluate and mix immediately Afterwards either follow the centrifugation protocol given after the NucleoBond Xtra purification protocol or follow the support protocol for the NucleoBond Finalizers in section 8 to eliminate the time consuming centrifugation steps for precipitation use of NucleoBond Finalizers is only recommended for vector sizes smaller than 50 kb The NucleoBond Final
58. or sufficient growth OD in the pres ence of an appropriate selective antibiotic Table 1 Second aliquots of the cleared lysate the flow through the combined washing steps buffers EQU and WASH and the eluate should be kept for further analysis by agarose gel electrophoresis Refer to Table 4 to choose a fraction volume yielding approximately 5 ug of plasmid DNA The volumes outlined in Table 4 refer to maximum yield per binding capacity of each column size used for the preparation Precipitate the nucleic acids by adding 0 7 volumes of isopropanol centrifuge the sample wash the pellet using 70 etha nol centrifuge again air dry for 10 minutes dissolve the DNA in 100 ul TE buffer pH 8 0 and run 20 ul on a 1 agarose gel The gel picture Figure 5 will help you to address the specific questions outlined in this section more quickly and efficiently Table 4 NucleoBond Xtra eluate volumes required for an analytical check Sample Purification step Volume required pl Midi Maxi Cleared lysate of protocol step 8 500 200 Column flow through after protocol step 8 500 200 Wash flow through II after protocol step 9 250 200 and 11 Eluate N after protocol step 12 100 100 56 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification Figure5 Analytical check of NucleoBond Xtra Midi purification samples Plasmid pUC18 bacterial strain E coli DH5a 20 ul of each precipitated sampl
59. ow copy plasmids NucleoBond Xtra kits are designed for isolation of high copy plasmids up to sev eral hundred copies cell as well as low copy plasmids lt 20 copies cell However when purifying low copy plasmids the cell mass and the lysis buffer volumes should be increased at least by factor 2 to provide enough DNA to utilize the columns bind ing capacity Table 3 shows recommended ODVs and the corresponding pairs of OD and culture volume for low copy plasmid cell cultures for detailed information on calculating ODV ODs X Vol refer to section 4 4 For higher yields it is advantageous to increase the cell culture and lysis buffer vol umes even more e g by factor 3 5 In this case additional lysis buffer can be or dered separately see section 9 2 for ordering information Furthermore a centrifuge should be used for lysate clarification instead of the provided NucleoBond Xtra column filters since their capacity for precipitate is limited Table 3 Suggested culture volumes for low copy plasmids Pellet Recommended culture volume for NucleoBond Wel Rec Xtra Kit weight ODV OD 500 OD 500 OD OD s00 OD 200 Midi 1 50 g 800 400 ml 200 ml 133 ml 100 ml 80 ml Maxi 4 50 g 2400 1200 ml 600 ml 400 ml 300 ml 240ml MACHEREY NAGEL 03 2006 Rev 01 13 Plasmid DNA Purification 4 6 Cell lysis The bacterial cell pellet is resuspended in buffer RES and lysed
60. r TRIS 15 ml 75 ml Plastic washer 5 10 10 5 10 Protocol For preparation of working solutions and storage conditions see section 5 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification 1 Kit contents continued NucleoBond Xtra NucleoBond Xtra Maxi Maxi Plus 10 preps 50 preps 100 preps 10 preps 50 preps Cat No 740414 10 740414 50 740414 100 740416 10 740416 50 Buffer RES 150 ml 750 ml 2x 750 ml 150 ml 750 ml Buffer LYS 150 ml 750 ml 2x 750 ml 150 ml 750 ml Buffer NEU 150 ml 750 ml 2x 750 ml 150 ml 750 ml 2x 1000 ml 5x 1000 ml 2 x 1000 ml Buffer EQU 500 ml 500 ml 500 ml 500 mi 1000 ml 3 x 1000 ml 1000 ml Buffer WASH 300 ml 500 ml 300 ml 500 ml Buffer ELU 180 ml 900 ml 2 x 900 ml 180 ml 900 ml RNase A lyophilized 10 mg 50 mg 2 x 50 mg 10 mg 50 mg NucleoBond Xtra 10 50 100 10 50 Maxi columns PAIGOS RONA ra 10 50 100 10 50 Maxi column filters NucleoBond g 10 50 Finalizer Large 30 ml Syringes 10 50 1 ml Syringes i 10 50 Buffer TRIS 15 ml 75 ml Plastic washer 5 10 10 5 10 Protocol For preparation of working solutions and storage conditions see section 5 6 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification Kit specifications NucleoBond Xtra kits are suitable for ultra fast purification of plasmids cosmids and very large constructs P1 constructs BACs PACs ranging from 3 kb up to 300 kb For preparation of
61. rch Zentrifugation bei 4 500 6 000 x g f r 10 min bei 4 C und entfernen Sie den berstand vollst ndig Hinweis Es k nnen auch gr ere Kulturvolumina verwendet werden z B wenn sich das Plasmid nicht wie ein typischer high copy Vektor verh lt f r weitere Informationen siehe Kapitel 4 4 In diesem Fall erh hen Sie die Volumina der Puffer RES LYS und NEU pro portional in den Schritten 4 5 und 7 Falls das Kulturvolumen mehr als doppelt so hoch ist wie das empfohlene verwenden Sie in Schritt 8 zur Lysatkl rung eine Zentrifuge an stelle des NucleoBond Xtra column filters MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 4 Resuspension Resuspendieren Sie das Zellpellet vollstandig in resuspension buffer RES RNase A durch Auf und Abpipettieren Wichtig fur eine effiziente Zelllyse ist dass keine Zellklumpen in der Suspension verbleiben Hinweis Erh hen Sie das Volumen des Puffers RES proportional falls mehr als die empfohlene Zellmasse eingesetzt wird f r Informationen zur optimalen Zelllyse siehe Kapitel 4 6 f r schwer zu lysierende Bakterienstamme siehe Kapitel 4 7 5 Zelllyse 6 Vor Gebrauch berpr fen Sie lysis buffer LYS auf ausgefallenes SDS Sollte ein weiBes Prazipitat sichtbar sein erwarmen Sie den Puffer f r einige Minuten auf 30 40 C bis das Pr zipitat komplett gel st ist Lassen Sie den Puffer auf Raum temperatur 20 25 C abk hlen
62. rge 11 Wash and dry DNA 70 ethanol 70 ethanol 70 ethanol 70 ethanol pellet 2 ml 2ml 5ml 5ml 15 000 x g 15 000 x g 5 min at RT 10 min at RT 5 10 min gt 3 x air 10 15 min gt 3 x air Appropriate Buffer TRIS Appropriate Buffer TRIS 12 Recon DNA volume of TE buffer 500 1000 pl volume of TE buffer 500 1000 ul NEW NEW NEW NEW Plasmid DNA Purification Table of contents 1 Kit contents 2 Kit specifications 3 About this user manual 4 NucleoBond Xtra plasmid purification system 4 1 Basic principle 4 2 NucleoBond Xtra anion exchange columns 4 3 Growth of bacterial cultures 4 4 Culture volume for high copy plasmids 4 5 Culture volume for low copy plasmids 4 6 Cell lysis 4 7 Difficult to lyse strains 4 8 Filtration and loading of the lysate 4 9 Washing of the column 4 10 Elution and concentration of plasmid DNA 4 11 Determination of DNA yield and quality 4 12 Convenient stopping points 5 Storage conditions and preparation of working solutions 6 Safety instructions risk and safety phrases 7 NucleoBond Xtra plasmid purification 7 1 High copy plasmid purification Midi Maxi English 7 2 High copy plasmid purification Midi Maxi German 7 3 High copy plasmid purification Midi Maxi French 7 4 Low copy plasmid purification Midi Maxi English 7 5 Low copy plasmid purification Midi Maxi German 7 6 Low copy plasmid purification Midi Maxi French 8 Concentration of Nu
63. row can be used In this case bacteria grow faster reach the stationary phase much earlier than in LB medium lt 12 h and higher cell masses can be reached However this does not necessarily yield more plasmid DNA Overgrowing a culture might lead to a higher percentage of dead or starving cells and the resulting plasmid DNA might be partially degraded or contaminated with chromosomal DNA To find the optimal culture condi tions the culture medium and incubation times have to be optimized for each host strain plasmid construct combination individually Cell cultures should be grown under antibiotic selection at all times to ensure plas mid propagation Without this selective pressure cells tend to lose a plasmid during cell division Since bacteria grow much faster without the burden of a high copy plasmid they take over the culture rapidly and the plasmid yield goes down regard less of the cell mass Table 1 gives information on concentrations of commonly used antibiotics Table 1 Information about antibiotics according to Maniatis Antibiotic Stock solution Storage Working concentration concentration Ampicillin 50 mg ml in H O 20 C 50 100 pg ml Chloramphenicol 34 mg ml in EtOH 20 C 25 170 ug ml Kanamycin 10 mg ml in H O 20 C 10 50 pg ml Streptomycin 10 mg ml in H O 20 C 10 50 pg ml Tetracycline 5 mg ml in EtOH 20 C 10 50 pg ml The E coli host strain mostly influences the quality of t
64. somal DNA The lysate should turn from a slimy viscous consistency to a low viscosity homogeneous suspension of an off white floccu late Immediately proceed with step 8 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 8 Clarification and loading Make sure to have a homogeneous suspension of the precipitate by inverting the tube 3 times directly before applying the lysate to the equilibrated NucleoBond Xtra column filter to avoid clogging of the filter The lysate is simultaneously cleared and loaded onto the column Refill the filter if more lysate has to be loaded than the filter is able to hold Allow the column to empty by gravity flow Alternative The precipitate can be removed by centrifugation at 25 000 x g for at least 10 min e g if more than double the recommended cell mass was used If the supernatant still contains suspended matter transfer it to a new tube and re peat the centrifugation preferably at higher speed or apply the lysate to the equilibrated NucleoBond Xtra column filter This clarification step is extremely important since residual precipitate may clog the NucleoBond Xtra column To load the column you can either apply the cleared lysate to the equilibrated filter or remove the unused filter beforehand Al low the column to empty by grav
65. t NEU dans les tapes 4 5 et 7 Si le volume de culture est plus du double du volume recommand il est plus judicieux de centrifuger pour clarifier le lysat tape 8 plut t que d utiliser le filtre NucleoBond Xtra 32 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 4 Resuspension Reprendre completement le culot cellulaire dans le tampon de resuspension resuspension buffer RES RNase A en pipetant les cellules par aspiration refoulement Important pour une lyse efficace des cellules il ne doit plus subsister d agregats en suspension Remarque Augmentez proportionnellement le volume du Tampon RES si vous avez utilis une masse cellulaire sup rieure a celle recommand e 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 a lyser 5 Lyse cellulaire Avant d utiliser le tampon LYS v rifiez que le SDS n a pas pr cipit Si un pr cipit blanc est visible chauffez le tampon quelques minutes a 30 40 C jusqu ce que le pr cipit soit compl tement dissout Ramenez le tampon a temp rature ambiante 20 25 C Rajoutez le tampon de lyse lysis buffer LYS la suspension M langez avec pr caution en inversant le tube 5 fois Ne pas utiliser de vortex sinon l ADN chromosomique se fractionnerait se detacherait des d bris cellulaires et contaminerait alors la susp
66. t a glance for a quick reference Each procedural step in the purification protocol is arranged like the following exam ple taken from section 7 1 5 Cell lysis Check lysis buffer LYS for precipitated SDS prior to use If a white precipitate is visible warm the buffer for several minutes at 30 40 C until precipitate is dis e solved completely Cool buffer down to room temperature 20 25 C Add lysis buffer LYS to the suspension Mix gently by inverting the tube 5 times Do not vortex as this will shear and release contaminating chromosomal DNA from cellular debris into the suspension Incubate the mixture at room temperature 20 25 C for 5 min Note Increase LYS buffer volume proportionally if more than the recommended cell mass is used see section 4 6 for information on optimal cell lysis If you are performing a Midi prep to purify plasmid DNA you will find volumes or incu bation times in the white boxes For Maxi preps please refer to the black boxes The name of the buffer buffer volume incubation times repeats or important han dling steps are emphasized in bold type within the instruction Additional notes or optional steps are printed in italic The exclamation point marks information and hints that are essential for a successful preparation In the example shown above you are asked to check the lysis buffer LYS prior to use and then to lyse the resuspended cell pellet in 8 ml of buffer LYS when p
67. ten 4 5 and 7 und verwenden in Schritt 8 zur Lysatkl rung eine Zentri fuge anstelle des NucleoBond Xtra column filters 42 Resuspension Resuspendieren Sie das Zellpellet vollst ndig in resuspension buffer RES RNase A durch Auf und Abpipettieren Wichtig f r eine effiziente Zellyse ist dass keine Zellklumpen in der Suspension verbleiben Hinweis Erh hen Sie das Volumen des Puffers RES proportional falls mehr als die empfohlene Zellmasse eingesetzt wird f r Informationen zur optimalen Zelllyse siehe Kapitel 4 6 f r schwer zu lysierende Bakterienst mme siehe Kapitel 4 7 MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi 5 Zelllyse Vor Gebrauch berpr fen Sie lysis buffer LYS auf ausgefallenes SDS Sollte ein wei es Pr zipitat sichtbar sein erw rmen Sie den Puffer f r einige Minuten auf 30 40 C bis das Pr zipitat komplett gel st ist Lassen Sie den Puffer auf Raum temperatur abk hlen 20 25 C Geben Sie lysis buffer LYS zu der Suspension Mischen Sie vorsichtig durch 5 maliges Invertieren Vortexen Sie nicht da dies zur Scherung der genomi schen DNA und zu deren Freisetzung aus den Zelltrummern in die Suspension f hren kann Inkubieren Sie die Mischung f r 5 min bei Raumtemperatur 20 25 C Hinweis Erh hen Sie das Volumen des Puffers LYS proportional falls mehr als die empfohlene Zellmasse eingesetzt wird f r Informationen zur optimalen Zelllyse siehe Kapitel
68. ter membrane Remove the NucleoBond Finalizer from the syringe pull out the plunger and reattach the NucleoBond Finalizer Press air through the NucleoBond Final izer with appropriate force while touching a tissue with the tip of the Nu cleoBond Finalizer to soak up ethanol Repeat this step at least two times until no more ethanol leaks from the Nu cleoBond Finalizer Note A new dry syringe can be used to speed up the procedure not provided Optional You can incubate the NucleoBond Finalizer for 10 minutes at 80 C to mini mize ethanol carry over However the final recovery may be reduced by over drying the DNA Elute DNA Remove the NucleoBond Finalizer from the syringe pull out the plunger of a 1 ml syringe and attach the NucleoBond Finalizer to the syringe outlet Note Refer to section 4 9 Figures 3 Midi or 4 Maxi to choose the appropriate vol ume of elution buffer Pipette 500 1000 ul of redissolving buffer TRIS 5 mM Tris HCl pH 8 5 or TE buffer into the syringe see section 4 10 Place the NucleoBond Finalizer outlet over a fresh collecting tube and elute plasmid DNA carefully by inserting the plunger 500 1000 jl 500 1000 pl 6 Remove the NucleoBond Finalizer from the syringe pull out the plunger and reattach the NucleoBond Finalizer to the syringe outlet Transfer the first eluate back into the syringe and elute into the same collecting tube a second tim
69. tively charged phosphate backbone of plasmid DNA to bind with high specificity Figure 1 DNA backbone e u ss er gt o silica __ spacer ANS CH e DNA backbone H Figure 1 lonic interaction of the positively charged methyl hydroxyethyl amino group with the negative phosphate oxygen of the DNA backbone In contrast to the widely used DEAE diethylaminoethanol group the hydroxy group of methyl hydroxyethyl amin can be in volved in additional hydrogen bonding interactions with the DNA Birnboim H C and Doly J 1979 Nucl Acids Res 7 1513 1523 MACHEREY NAGEL 03 2006 Rev 01 9 Plasmid DNA Purification Due to a specialized manufacturing process that is strictly controlled and monitored the NucleoBond Xtra silica beads are uniform in diameter and contain particularly large pores These special properties allow optimized flow rates and sharp well defined elution profiles NucleoBond Xtra can separate distinct nucleic acid species from each other and from proteins carbohydrates and other unwanted cellular com ponents over an exceptionally broad range of salt concentrations Figure 2 All contaminants from proteins to RNA are washed from the column the positive charge of the resin is neutralized by a pH shift to slightly alkaline conditions and pure plasmid DNA is eluted in a high salt elution buffer The purified nucleic acid products are suitable for use in the most demanding mo lecu
70. to section 9 2 for ordering information By using sufficient amounts of lysis buffer lysis time can be limited to 3 4 minutes and should not exceed 5 minutes Prolonged exposure to alkaline conditions can ir reversibly denature and degrade plasmid DNA and liberate contaminating chromo somal DNA into the lysate 4 7 Difficult to lyse strains For plasmid purification of e g Gram positive bacteria or strains with a more resistant cell wall it might be advantageous to start the preparation with a lysozyme treatment 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 14 MACHEREY NAGEL 03 2006 Rev 01 Plasmid DNA Purification 4 8 Filtration and loading of the lysate After the alkaline lysis the sample has to be cleared from cell debris and precipitate to ensure high plasmid purity and a fast column flow rate This is achieved by passing the solution through a NucleoBond Xtra column filter which is provided already inserted into the NucleoBond Xtra column Midi Maxi NucleoBond Xtra column filter NucleoBond Xtra Ni NA The NucleoBond Xtra column filters are designed to eliminate the centrifugation step after alkaline lysis They are pre wet during column equilibration and allow a time saving simultaneous clearing of bacterial lysate and loading of the Nu cl
71. ucleoBond Xtra Maxi Plus Option D terminez le rendement d ADN plasmidique par spectrophotom trie UV afin d ajuster la concentration de l ADN l tape 15 et calculez le taux de r cup ration apr s l tape de pr cipitation 13 Pr cipitation Ajoutez l isopropanol temp rature ambiante pour pr cipiter l ADN plasmidique lu Vortexez et laissez le m lange reposer pendant 2 minutes Centrifugez 5 000 x g pendant 215 min lt temp rature ambiante de pr f rence 15 000 x g pendant 30 min 4 C Eliminez pr cautionneusement le surnageant 14 36 Lavage et s chage du culot d ADN Ajoutez l thanol 70 a temp rature ambiante sur les culots et centrifugez 5 000 x g de pr f rence 15 000 x g pendant 5 min temp rature ambiante 20 25 C MACHEREY NAGEL 03 2006 Rev 01 NucleoBond Xtra Midi Maxi Eliminez pr cautionneusement l thanol compl tement avec une pipette Laissez s cher le culot a temp rature ambiante 20 25 C Remarque l ADN plasmidique peut tre plus difficile dissoudre si le culot est tr s sec 15 Reconstitution de ADN Dissoudre le culot d ADN dans un volume ad quat de tampon TE ou d eau distill e st rile En fonction du type de tube utilis pour la centrifugation dissoudre par aspiration refoulement ou par rotation constante dans un volume appropri
72. veral times after elution and collecting every single droplet to minimize the dead volume 100 0 50 BR 80 7 0 40 5 zL 897 7 9 30 5 Recovery gt BE j 8 40 0 20 8 Concentration 2 ec 20 0 10 O 0 j j j 0 00 300 500 700 900 1100 Elution Volume pl Figure 3 Concentration of NucleoBond Xtra Midi eluates using NucleoBond Finalizer Final recovery and concentration of 250 ug plasmid DNA 8 kbp loaded onto a NucleoBond Finalizer and eluted two fold with varying volumes of low salt buffer 100 2 50 80 2 00 3 gt B Tits Recovery gt 8 404 1 00 Conceniration 2 a 20 0 50 O 0 i i i 0 00 300 500 700 900 1100 Elution Volume pl Figure 4 Concentration of NucleoBond Xtra Maxi eluates using NucleoBond Finalizer Large Final recovery and concentration of 1000 ug plasmid DNA 8 kbp loaded onto a NucleoBond Finalizer and eluted two fold with varying volumes of low salt buffer NucleoBond Finalizer is designed to hold a maximum of 500 ug DNA and is therefore ideally suited for the use in combination with a NucleoBond Xtra Midi kit NucleoBond Xtra Maxi eluates are easily concentrated with a NucleoBond Fi nalizer Large which is able to bind up to 2000 ug plasmid DNA Optimal recoveries are achieved with both NucleoBond Finalizers by using 1000 ul of elution buffer MACHEREY NAGEL 03 200
73. working solutions and storage conditions see section 5 NucleoBond Xtra columns are polypropylene columns containing Nu cleoBond Xtra silica resin packed between two inert filter elements The columns are available in Midi and Maxi sizes with typical maximum DNA yields of 250 ug and 1000 ug respectively All NucleoBond Xtra columns are resistant to organic solvents such as al cohol chloroform and phenol and are also suitable for buffers containing de naturing agents like formamide urea or common detergents like Triton X 100 or NP 40 NucleoBond Xtra silica resin can be used over a wide pH range pH 2 5 8 5 and can remain in contact with buffers for several hours without any change in its chromatographic properties The NucleoBond Xtra column filters are specially designed depth filters that fit into the NucleoBond Xtra columns The filters are inserted ready to use in the NucleoBond Xtra columns and allow a time saving simultaneous clearing of bacterial lysate and loading of cleared lysate onto the NucleoBond Xtra column Furthermore the use of the column filters avoids the time consuming centrifugation step for lysate clearing The NucleoBond Xtra column filters allow complete removal of precipitate even with large lysate volumes without clogging and avoid shearing of large DNA constructs such as PACs or BACs by the gentle depth filter effect The NucleoBond Xtra Midi Plus and NucleoBond Xtra Maxi Plus kits ad

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