NucleoBond® PC - MACHEREY
Contents
1. 5 Binding Load the cleared lysate from step 4 onto the NucleoBond Column Allow the column to empty by gravity flow Optional You may want to save all or part of the flow through for analysis 6 Washing Wash the column with Buffer N3 Repeat as indicated Discard flow through 2x 18 mL 2x50 mL 7 Elution Elute the plasmid DNA with Buffer N5 Preheating Buffer N5 to 50 C prior to elution may improve yields for high molecular weight constructs such as BACs We recommend precipitating the eluate as soon as possible step 8 Nevertheless the eluate can be stored in closed vials on ice for several hours In this case the eluate should be preheated to room temperature before the plasmid DNA is precipitated 15 mL 25 mL Optional Determine plasmid yield by UV spectrophotometry in order to adjust the desired concentration of DNA step 10 34 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification Maxi Mega AX 500 BAC 100 AX 2000 8 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Mix carefully and centrifuge at 15 000 x g for 30 min at 4 C Carefully discard the supernatant 11 mL 18 mL 9 Wash and dry DNA pellet Add room temperature 70 ethanol to the pellet Vortex briefly and centrifuge at 15 000 x g for 10 min at room temperature 18 25 C 5mL 7mL Carefully remove ethanol from the tube w2. Nucleic acid did not precipitate Check volumes of precipitating solvent making sure to use at least 0 7 volumes of isopropanol and centrifuge for longer periods of time Nucleic acid pellet does not resuspend in buffer Pellet was over dried Try dissolving at higher temperatures for a longer period of time e g 2 h at 37 C or overnight at RT best under constant spinning 3D shaker Residual salt or organic solvent in the pellet Wash the pellet with additional low viscosity organic solvent 70 ethanol or increase the resuspension buffer volume Nucleic acid pellet is Opaque or white instead of clear and glassy Salt has co precipitated with the pellet Use room temperature isopropanol and check isopropanol purity Do not precipitate by allowing the eluate to drip directly from the column into a tube containing isopropanol Add isopropanol only after eluate has been collected Try resuspending the pellet in Buffer N2 and reload onto the NucleoBond Column Be sure to wash the column several times with Buffer N2 before loading the redissolved pellet onto the column MACHEREY NAGEL 11 2011 Rev 09 41 Plasmid DNA purification Problem Possible cause and suggestions DNA is contaminated with cellular debris or genomic DNA due to inefficient Iysis BR Reduce the culture volume or increase the amount of Buffer Purified plas S1 S2 and S3 used during the lysis steps mid does not perf
3. Plasmid DNA purification User manual NucleoBond PC 20 NucleoBond PC 100 NucleoBond PC 500 NucleoBond BAC 100 NucleoBond PC 2000 NucleoBond PC 10000 November 2011 Rev 09 MACHEREY NAGEL KT Plasmid DNA Purification Mini Midi Maxi Mega Giga Protocol at a glance Rev 09 Mini Midi Maxi Mega Giga AX 20 AX 100 AX 500 AX 2000 AX 10000 1 Cultivate and harvest 4 500 6 000 xg 4 500 6 000xg 4 500 6 000xg 4 500 6 000xg 4 500 6 000 x g es 4 C 15 min 4 C 15 min 4 C 15 min 4 C 15 min 4 C 15 min bacterial cells 2 Cell Iysis High copy low copy Buffer S1 0 4 mL 0 8 mL 4mL 8 mL 12 mL 24mL 45 mL 90 mL 120 mL Buffer S2 0 4 mL 0 8 mL 4mL 8 mL 12 mL 24 mL 45 mL 90 mL 120 mL m RT lt 5 min RT lt 5 min RT lt 5 min RT lt 5 min RT lt 5 min Buffer S3 0 4 mL 0 8 mL 4mL 8 mL 12 mL 24 mL 45 mL 90 mL 120 mL 7 0 C 5 min 0 C 5 min 0 C 5 min 0 C 5 min 0 C 5 min 3 Equilibration of the column Buffer N2 Buffer N2 Buffer N2 Buffer N2 Buffer N2 1 1 mL 2 5 mL 6 mL 20 mL 100 mL Bi 4 Clarification j f j j of the Iysate Centrifugation Folded Filter Folded Filter Folded Filter Folded Filter or centrifugation or centrifugation or centrifugation or centrifugation 12 000 x g 12 000 x g 12 000 x g 12 000 x g 12 000 x g 15 min 25 min 40 min 50 min 60 min 5 Binding Load cleared Load cleared Load cleared Load cleared Load cle
4. 11 2011 Rev 09 15 Plasmid DNA purification 3 5 Filtration of the Iysate After alkaline Iysis the solution has to be cleared from precipitated protein and cell debris in order to prevent clogging of the NucleoBond Columns There are the following three options 1 Use the NucleoBond Folded Filters provided with the kits The gentle filtration prevents shearing of plasmids and large constructs such as cosmid PAC or BAC DNA For NucleoBond PC 100 500 2000 BAC 100 place one filter in a funnel of appropriate size For NucleoBond PC 10000 put a folded filter of type 1 into a folded filter of type 2 and place the combination in a funnel The two types of filters differ in pore size to allow a fast and complete removal of large amounts of precipitate Wet the filter s with a few drops of Equilibration Buffer N2 and load the bacterial lysate onto the wet filter s Either collect the flow through in a separate vessel or position funnel and filter directly Figure 2 Correct use of on top of the NucleoBond Column to clear and load the NucleoBond Folded the lysate in one time saving step see Figure 2 Filter NucleoBond Column placed in a Plastic Washer 2 Use NucleoBond Bottle Top Filters for a very fast and complete vacuum assisted filtration Two different types of filters can be ordered separately see ordering information for NucleoBond PC 2000 Type 1 or NucleoBond PC 10000 Type 2 Attach a bottle
5. from step 4 onto the NucleoBond Column Allow the column to empty by gravity flow Optional You may want to save all or part of the flow through for analysis Washing Wash the column with Buffer N3 Repeat as indicated Discard flow through 2x2mL 12 mL Elution Elute the plasmid DNA with Buffer N5 Preheating Buffer N5 to 50 C prior to elu tion may improve yields for high molecular weight constructs such as BACs We recommend precipitating the eluate as soon as possible step 8 Nevertheless the eluate can be stored in closed vials on ice for several hours In this case the eluate should be preheated to room temperature before the plasmid DNA is precipitated 1mL 5 mL Optional Determine plasmid yield by UV spectrophotometry in order to adjust the desired concentration of DNA step 10 MACHEREY NAGEL 11 2011 Rev 09 31 Plasmid DNA purification Mini Midi AX 20 AX 100 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Mix carefully and centrifuge at 15 000 x g for 30 min at 4 C Carefully discard the supernatant 0 75 mL 3 5 mL 10 Wash and dry DNA pellet Add room temperature 70 ethanol to the pellet Vortex briefly and centrifuge at 15 000 x g for 10 min at room temperature 18 25 C 500 uL 2mL Carefully remove ethanol from the tube with a pipette tip Allow the pellet t
6. g for 15 min at 4 C 2 Cell lysis Carefully resuspend the pellet of bacterial cells in Buffer S1 RNase A Please see section 6 3 regarding difficult to lyse strains Add Buffer S2 to the suspension Mix gently by inverting the tube 6 8 times Incubate the mixture at room temperature 18 25 C for 2 3 min max 5 min Do not vortex as this will release contaminating chromosomal DNA from cellular debris into the suspension 0 4 mL 4mL 12 mL Add pre cooled Buffer S3 4 C to the suspension Immediately mix the lysate gently by inverting the flask 6 8 times until a homogeneous suspension containing an off white flocculate is formed Incubate the suspension on ice for 5 min 24 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification Mini Midi Mai AX 20 AX 100 AX 500 Equilibration of the column Equilibrate a NucleoBond AX 20 Mini AX 100 Midi or AX 500 Maxi Column with Buffer N2 Allow the column to empty by gravity flow Discard flow through 1 0 mL 2 5 mb Clarification of the lysate Note Complete removal of precipitated protein and cell debris is essential to avoid clogging of the NucleoBond Column NucleoBond PC 20 Centrifuge the lysate at gt 12 000 x g for 5 10 minutes at room temperature or better 4 C NucleoBond PC 100 500 Place a NucleoBond Folded Filter in a funnel of appropriate size Wet the
7. hydroxide 0 5 2 0 x Xi R 36 38 S 26 Natriumacetat 0 5 2 0 37 39 45 RNase A RNase A Iyophilized x Xn R 42 43 S 22 24 RNase A lyophilisiert Risk phrases R10 Flammable Entz ndlich R 36 38 Irritating to eyes and skin Reizt die Augen und die Haut R 42 43 May cause sensitization by inhalation and skin contact Sensibilisierung durch Einatmen und Hautkontakt m glich Safety phrases S22 Do not breathe dust Staub nicht einatmen S 24 Avoid contact with the skin Ber hrung mit der Haut vermeiden S 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice Bei Ber hrung mit den Augen sofort gr ndlich mit Wasser absp len und Arzt konsultieren S 37 39 Wear suitable gloves and eye face protection Bei der Arbeit geeignete Schutzhandschuhe und Schutzbrille Gesichtsschutz tragen S45 In case of accident or if you feel unwell seek medical advice immediately show the label where possible Bei Unfall oder Unwohlsein sofort Arzt zuziehen wenn m glich dieses Etikett vorzeigen Hazard labeling not necessary if quantity per bottle below 25g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet MACHEREY NAGEL 11 2011 Rev 09 19 Plasmid DNA purification 5 2 GHS classification Only harmful features do not need to b
8. routine purification of low copy plasmids The NucleoBond Buffer Set I can be used in connection with NucleoBond PC kits for the isolation of low copy plasmids In this combination it is sufficient for NucleoBond PC 500 kit REF 740574 10 preparations low copy plasmid purification NucleoBond PC 100 kit REF 740573 20 preparations low copy plasmid purification NucleoBond PC 20 kit REF 740571 100 100 preparations low copy plasmid purification In connection with NucleoBond AX Columns the NucleoBond Buffer Set I can be used for the isolation of high copy plasmids In this combination it is sufficient for NucleoBond PC 500 Columns REF 740531 5 preparations high copy plasmid purification NucleoBond PC 100 Columns REF 740521 10 preparations high copy plasmid purification NucleoBond PC 20 Columns REF 740511 50 preparations high copy plasmid purification The NucleoBond BAC 100 kit is recommended for the isolation of low copy plasmids and contains sufficient buffer to perform 10 maxi preps The kit contains BAC 100 columns which can bind up to 500 ug of plasmid DNA Typically yields are 10 100 ug from 500 mL fermentation broth depending on copy number and size of constructs also see section 6 for further information regarding the growing of bacterial cultures The protocol for the isolation of low copy plasmids using the NucleoBond BAC 100 kit can be found in section 7 5 MACHEREY NAGEL
9. run 20 uL on a 1 agarose gel Table 4 NucleoBond PC volumes required for an analytical check Sample Purification step Volume required pL PC 100 PC 500 PC 2000 PC 10000 Cleared Iysate after protocol step 4 600 499 200 B Column flow ll through 600 400 300 200 after protocol step 5 Wash flow through lli Ti ar Erelocel ste 500 300 200 100 IV Elnale 300 200 100 100 after protocol step 7 The exemplary gel picture see Figure 4 will help you to address the specific questions outlined in the following section more quickly and efficiently It shows for example the dominant plasmid bands which should only be present in the eluate and in the Iysate before loading to proof plasmid production in your cell culture lane 1 Plasmid DNA found in the wash fractions however narrows down the problem to wrong or bad wash buffers e g wrong pH buffer components precipitated evaporation of liquid due to wrong storage RNA might be visible as a broad band at the bottom of the gel for the Iysate and the lysate flow through samples lane 1 and 2 It might also occur in the wash fraction but must be absent in the eluate Genomic DNA should not be visible at all but would show up in the gel slot or right below indicating e g too harsh lysis conditions MACHEREY NAGEL 11 2011 Rev 09 37 Plasmid DNA purification M Marker Hindlll 1 cleared lysate ccc linear an
10. we can not grant any guarantees regarding selection efficiency or operation 46 MACHEREY NAGEL 11 2011 Rev 09
11. 00 20 syringe sets PC 500 EF NucleoBond Xtra Midi Midi EF NucleoBond Finalizer Large 740418 20 20 large filters for use with NucleoBond PC 2000 PC 2000 EF 2 syringe sets NucleoBond Xtra Maxi Maxi EF NucleoBond Finalizer Large Plus 740419 20 20 large filters for use with NucleoBond PC 2000 PC 2000 EF 20 syringe sets NucleoBond Xtra Maxi Maxi EF NucleoBond Folded Filters 740561 5 for NucleoBond AX 100 Columns MACHEREY NAGEL 11 2011 Rev 09 43 Plasmid DNA purification Product REF Pack of NucleoBond Folded Filters XL 740577 50 for NucleoBond AX 500 2000 BAC 100 Columns NucleoBond Bottle Top Filters Type 1 740547 5 5 for NucleoBond AX 2000 Columns NucleoBond Bottle Top Filters Type 2 740553 5 5 for NucleoBond AX 10000 Columns NucleoBond Buffer Set 740601 1 set Buffer S1 740516 1 500 mL Buffer S2 740517 1 500 mL Buffer S3 740518 1 500 mL Buffer N2 740527 1 500 mL Buffer N3 740528 1 1000 mL Buffer N5 740529 1 500 mL NucleoBond Rack Small 740562 1 for NucleoBond AX 20 Columns NucleoBond Rack Large 740563 1 for NucleoBond AX 100 500 2000 and 10000 Columns RNase A lyophilized 740505 50 50 mg 740505 100 mg Visit www mn net com for more detailed product information 8 4 References Birnboim H C and Doly J 1979 Nucl Acids Res 7 1513 1523 44 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 8 5 Produ
12. 80 Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen P 302 352 IF ON SKIN Wash with plenty of soap and water Bei Kontakt mit der Haut Mit viel Wasser und Seife waschen 20 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification P 3044341 P 305 351 388 P 332 313 P 333 313 P 337 313 P 342 311 P 363 P 403 235 IF INHALED If breathing is difficult remove to fresh air and keep at rest ina position comfortable for breathing Bei Einatmen Bei Atembeschwerden an die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert IF IN EYES Rinse continuously with water for several minutes Remove contact lenses if present and easy to do continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter aussp len IF skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltender Augenreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER or doc tor physician Bei Symptomen der Atemwege Giftinformationszentrum oder Arzt anrufen Wash contaminated clothing befo
13. AGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications 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 to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this pr
14. Nase A lyophilized 15 mg 2x25 mg 2x 40 mg 3 x 50 mg NucleoBond AX 500 10 25 50 100 Columns NucleoBond Folded Filters XL je 23 ap 100 Plastic Washers 5 10 10 10 User Manual 1 1 1 1 For preparation of working solutions and storage conditions see section 4 MACHEREY NAGEL 11 2011 Rev 09 T Plasmid DNA purification 1 1 Kit contents continued NucleoBond PC 2000 NucleoBond PC 10000 5 preps 5 preps REF 740576 740593 Resuspension Buffer S1 250 mL 750 mL Lysis Buffer S2 250 mL 750 mL Neutralization Buffer S3 250 mL 750 mL Equilibration Buffer N2 125 mL 500 mL 125 mL Wash Buffer N3 2 x 250 mL 1000 mL 125 mL Elution Buffer N5 200 mL 500 mL 120 mL RNase A lyophilized 25 mg 80 mg NucleoBond 5 AX 2000 Columns NucleoBond 5 AX 10000 Columns NucleoBond 5 Folded Filters XL NucleoBond 10 Folded Filters Type 1 NucleoBond 10 Folded Filters Type 2 Plastic Washers 5 User Manual 1 1 For preparation of working solutions and storage conditions see section 4 8 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 1 1 Kit contents continued NucleoBond BAC 100 10 preps REF 740579 Resuspension Buffer S1 2x150mL Lysis Buffer S2 2x 150 mL Neutralization Buffer S3 2x 150 mL Equilibration Buffer N2 70 mL Wash Buffer N3 2 x 250 mL Elution Buffer N5 150 mL RNase A lyophilized 2x 15mg NucleoBond BAC 100 Columns 10 NucleoBond Folded Filters XL 10
15. Plastic Washers 5 User Manual 1 For preparation of working solutions and storage conditions see section 4 MACHEREY NAGEL 11 2011 Rev 09 9 Plasmid DNA purification 1 2 Reagents and equipment to be supplied by user Reagents Isopropanol room temperatured 70 ethanol room temperatured Ice Buffer for reconstitution of DNA e g TE buffer or sterile H O Equipment Standard microbiological equipment for growing and harvesting bacteria e g inoculating loop culture tubes and flasks 37 C shaking incubator and centrifuge with rotor and tubes or bottles for harvesting cells Funnels to hold the NucleoBond Folded Filters for lysate filtration NucleoBond PC 100 500 2000 10000 BAC 100 NucleoBond Rack see ordering information or equivalent holder Refrigerated centrifuge capable of reaching 15 000 x g with rotor for the appropriate centrifuge tubes or bottles Centrifugation tubes or vessels with suitable capacity for the volumes specified in the respective protocol 10 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 2 Introduction 2 1 Properties NucleoBond AX is a patented silica based anion exchange resin developed by MACHEREY NAGEL for routine separation of different classes of nucleic acids NucleoBond AX Resin forms the basis for the entire line of nucleic acid purification products presented in this User Manual NucleoBond AX Resin consists of hydrophi
16. age blocked Check cleared lysate for precipitates especially if the lysate was stored for a longer time before loading If necessary clear the lysate again by filtration Lysate was not completely cleared Centrifuge at higher speed for a longer period of time or use additional NucleoBond Folded Filters to clear the lysate Lysis treatment was too harsh Be sure not to incubate the lysate in Buffer S2 for more than 5 min Cellular DNA Overzealous mixing during lysis allowed genomic DNA to shear off or RNA con into the lysis buffer tamination of If the lysate is too viscous to mix properly or gently reduce plasmid DNA culture volumes RNase digestion was inefficient RNase was not added to Buffer S1 or stored too long Add new RNase to Buffer S1 See ordering information section 8 3 40 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification Problem Possible cause and suggestions Pellet was lost Handle the precipitate with care Decant solutions carefully Measure DNA yield in Buffer N5 in order to calculate the potential plasmid DNA that should be recovered after precipitation No nucleic Pellet did not resuspend in buffer acid pellet Again handle the pellet with care Especially if the DNA was formed after precipitation precipitated in a gt 15 mL tube the pellet may be smeared over the wall of the tube Dissolve DNA with an appropriate volume of TE buffer by rolling the tube for at least 30 min
17. ared lysate onto the lysate onto the lysate onto the lysate onto the lysate onto the column column column column column 6 Washing Buffer N3 Buffer N3 Buffer N3 Buffer N3 Buffer N3 High copy High copy High copy High copy High copy 2x1 5mL 10 mL 32 mL 2x35 mL 2x 100 mL j Low copy Low copy Low copy Low copy d 2x2mL 12 mL 2x18 mL 2 x 50 mL 7 Elution Buffer N5 Buffer N5 Buffer N5 Buffer N5 Buffer N5 1 mL 5mL 15 mL 25 mL 100 mL 8 Precipitation ii Isopropanol Isopropanol Isopropanol Isopropanol Isopropanol 0 75 mL 3 5 mL 11 mL 18 mL 70 mL j gt 15 000 x g gt 15 000 x g gt 15 000 x g gt 15 000 x g gt 15 000 x g e gt 4 C 30 min 4 C 30 min 4 C 30 min 4 C 30 min 4 C 30 min 9 Wash and dry i DNA pellet 70 ethanol 70 ethanol 70 ethanol 70 ethanol 70 ethanol 500 uL 2 mL 5 mL 7mL 10 mL a 15 000 xg gt 15 000 x g 15 000 x g 15 000 x g 15 000 x g cs RT 10 min RT 10 min RT 10 min RT 10 min RT 10 min 5 10 min 5 10 min 10 20 min 30 60 min 30 60 min 10 Reconstitute z DNA Appropriate Appropriate Appropriate Appropriate Appropriate volume of TE volume of TE volume of TE volume of TE volume of TE MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Tel 49 24 21 969 270 Fax 49 24 21 969 199 tech bio mn net com www mn net com Plasmid DNA purification Table of contents 1 Components 1 1 Kit contents 1 2 Reagents and equipment to be supplied by user 2 Introduct
18. ary Sample lysate is too viscous Watch maximal volumes and pellet wet weights given in the manual Otherwise filtration of the lysate and flow rate of the column will be insufficient No or low aa DNA Column overloaded with nucleic acids yie Use a larger column or purify excess nucleic acids on a new column Refer to the recommended culture volumes listed in the table at the beginning of each protocol 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 appropriate antibiotic concentration to plates and media Alkaline lysis was inefficient If culture volume or pellet weight is too high alkaline lysis be comes inefficient Refer to the recommended culture volumes listed in Table 2 section 6 1 Lysate incorrectly prepared After storage below 20 C SDS in Buffer S2 may precipitate causing inefficient lysis Check BufferS2 for precipitates before use and preheat the bottle to 30 40 C if necessary in order to redissolve SDS MACHEREY NAGEL 11 2011 Rev 09 39 Plasmid DNA purification Problem Possible cause and suggestions Sample is too viscous Do NOT attempt to purify Iysate prepared from a culture volume larger than recommended for any given column size Increasing culture volumes not only block the column but can also reduce yields due to inefficient lysis Column is Precipitates occur during stor
19. ation Buffer N2 25 mL 125 mL Wash Buffer N3 3 x 30 mL 3x 125 mL Elution Buffer N5 32 mL 120 mL RNase A lyophilized 2 5 mg 2x2 5mg NucleoBond AX 20 Columns 20 100 Plastic Washers 10 10 User Manual 1 1 For preparation of working solutions and storage conditions see section 4 MACHEREY NAGEL 11 2011 Rev 09 5 Plasmid DNA purification 1 1 Kit contents continued NucleoBond PC 100 20 preps 100 preps REF 740573 740573 100 Resuspension Buffer S1 100 mL 500 mL Lysis Buffer S2 4x25mL 500 mL Neutralization Buffer S3 100 mL 500 mL Equilibration Buffer N2 70 mL 2 x 150 mL Wash Buffer N3 250 mL 1000 mL 125 mL Elution Buffer N5 120 mL 3 x 200 mL RNase A lyophilized 10 mg 50 mg NucleoBond AX 100 Columns 20 100 NucleoBond Folded Filters 20 100 Plastic Washers 10 10 User Manual 1 1 For preparation of working solutions and storage conditions see section 4 6 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 1 1 Kit contents continued NucleoBond PC 500 10 preps 25 preps 50 preps 100 preps REF 740574 740574 25 740574 50 740574 100 Resuspension Buffer S1 150 mL 2x200 mL 2x400 mL 3x500 mL Lysis Buffer S2 150 mL 400 mL 2x400 mL 3x500 mL Neutralization Buffer S3 150 mL 400 mL 2x400 mL 3x500 mL Equilibration Buffer N2 70 mL 200 mL 2 x 200 mL 500 mL 200 mL Wash Buffer N3 2 x 250 mL 1000 mL 2x1000 mL 3x 1000 mL 500 mL Elution Buffer N5 200 mL 500 mL 2x500 mL 3x500 mL 200 mL R
20. cation kits contain NucleoBond Columns appropriate buffers and RNase A Kits are available for each column size Mini PC 20 Midi PC 100 Maxi PC 500 BAC 100 Mega PC 2000 and Giga PC 10000 The protocols are suitable for purifying most plasmids ranging from 3 10 kbp cosmids from 10 50 kbp and very large constructs P1 constructs BACs PACs up to 300 kbp NucleoBond Columns are polypropylene columns containing NucleoBond AX Silica Resin packed between two inert filter elements The columns are available in several sizes to accommodate a wide range of purification needs see Table 1 Table 1 NucleoBond Column binding capacities NucleoBond Column Binding capacity AX 20 20 ug AX 100 100 ug AX 500 500 ug BAC 100 100 ug AX 2000 2mg AX 10000 10 mg All NucleoBond Columns are resistant to organic solvents such as alcohol chloroform and phenol and are free of DNase and RNase MACHEREY NAGEL 11 2011 Rev 09 13 Plasmid DNA purification NucleoBond AX Resin can be used over a wide pH range from pH 2 5 8 5 and can remain in contact with buffers for up to three hours without any change in its chromatographic properties After three hours nucleic acids will begin to elute at increasingly lower salt concentrations Normally the resin remains functional in buffers containing up to 2 M salt It remains intact in the presence of denaturing agents like formamide urea or common detergents s
21. ct use restriction warranty NucleoBond PC BAC kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN V TRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DI
22. d goggles Storage conitions All kit components can be stored at room temperature 18 25 C and are stable for up to one year Before you start any NucleoBond plasmid DNA purification prepare the following Dissolve the lyophilized RNase A by the addition of 1 mL Resuspension Buffer S1 Wearing gloves is recommended Pipette up and down until the RNase A is dissolved completely Transfer the RNase A solution back to the bottle containing Buffer S1 and shake well Indicate date of RNase A addition The final concentration of RNase A is 100 g mL Buffer S1 Store Buffer S1 with RNase A at 4 C The solution will be stable at this temperature for at least 6 months Lysis Buffer S2 should be stored at room temperature 18 25 C since the containing SDS may precipitate at temperatures below 20 C If precipitation occurs incubate the bottle for several minutes at about 30 40 C and mix well until the precipitate is redissolved 18 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 5 Safety instructions The following components of the NucleoBond PC kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section 5 1 Risk and safety phrases Component Hazard contents Hazard Risk Safety symbol phrases phrases Inhalt Gefahrstoff Gefahrstoff R S tze S S tze symbol N2 N3 N5 Buffer salts ethanol 5 20 x R 10 Puffersalze Ethanol 5 20 S2 Sodium
23. d centrifuge at 15 000 x g for 10 min at room temperature 18 25 C 7mL 10 mL Carefully remove ethanol from the tube with a pipette tip Allow the pellet to dry at room temperature 18 25 C no less than the indicated time Drying for longer periods of time will not harm the quality of plasmid DNA but over drying may render the DNA less soluble 30 60 min 30 60 min 10 Reconstitute DNA Dissolve pellet in an appropriate volume of buffer TE or sterile deionized HO Depending on the type of centrifugation tube dissolve under 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 MACHEREY NAGEL 11 2011 Rev 09 29 Plasmid DNA purification 7 4 Low copy plasmid purification Mini Midi Mini Midi AX 20 AX 100 1 Cultivate and harvest bacterial cells Harvest bacteria from an LB culture by centrifugation at 4 500 6 000 x g for 15 min at 4 C 2 Cell lysis Carefully resuspend the pellet of bacterial cells in Buffer S1 RNase A Please see section 6 3 regarding difficult to lyse strains 0 8 mL 8 0 mL Add Buffer S2 to the suspension Mix gently by inverting the tube 6 8 times Incubate the mixture at room temperature 18 25 C for 2 3 min max 5 min Do not vortex as this will release contaminating chromosomal DNA from cellular
24. d oc structure of the plasmid degraded RNA Il lysate flow through no plasmid DNA but degraded RNA bed ww 3 Ill wash flow through no plasmid DNA or u residual RNA 4 IV eluate pure plasmid DNA 5 EcoRI restriction linearized form of plasmid M I er Zu u ett Figure 4 Exemplary analytical check of NucleoBond PC 500 purification samples Plasmid pUC18 bacterial strain E coli DH5a 20 HL of each precipitated sample has been analyzed on a 1 TAE agarose gel Equal amounts of plasmid DNA before lane 1 and after lane 4 purification using NucleoBond PC 500 are shown with a recovery of gt 90 38 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification Problem Possible cause and suggestions SDS or other precipitates are present in the sample Load the S1 S2 S3 lysate sample onto the NucleoBond Column immediately after finishing the initial lysis steps SDS and cell debris are removed by filtration with NucleoBond Folded Filters NucleoBond Bottle Top Filters but if the cleared lysate is stored on ice for a longer period new precipitate may appear If precipitate is visible it is recommended to filter the lysate again immediately before loading it onto the NucleoBond Column PH or salt concentrations of buffers are too high Especially if the customer prepares additional buffer it is recommended to thoroughly check the pH of each buffer Adjust pH or prepare new buffers if necess
25. debris into the suspension 0 8 mL 8 0 mL Add pre cooled Buffer S3 4 C to the suspension Immediately mix the lysate gently by inverting the flask 6 8 times until a homogeneous suspension containing an off white flocculate is formed Incubate the suspension on ice for 5 min 0 8 mL 8 0 mL 3 Equilibration of the column Equilibrate a NucleoBond AX 20 Mini AX 100 Midi Column with Buffer N2 Allow the column to empty by gravity flow Discard flow through 1mL 2 5 mL 30 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification Mini Midi AX 20 AX 100 Clarification of the Iysate Note Complete removal of precipitated protein and cell debris is essential to avoid clogging of the NucleoBond Column NucleoBond PC 20 Centrifuge the lysate at gt 1 2 000 x g for 5 10 minutes at room temperature or better 4 C NucleoBond PC 100 500 Place a NucleoBond Folded Filter in a funnel of appropriate size Wet the filter with a few drops of Buffer N2 and load the bacterial lysate onto the wet filter Either collect the flow through in a separate vessel and proceed with step 5 OR position funnel and filter directly on top of the NucleoBond Column to clear and load the lysate in one time saving step skip step 5 See section 3 5 for more information on how to set up the funnel and filter and for alternative clarification methods like centrifugation Binding Load the cleared lysate
26. e NucleoBond Column to clear and load the lysate in one time saving step skip step 5 See section 3 5 for more information on how to set up the funnel and filter and for alternative clarification methods like centrifugation Binding Load the cleared lysate from step 4 onto the NucleoBond Column Allow the column to empty by gravity flow Optional You may want to save all or part of the flow through for analysis Washing Wash the column with Buffer N3 Repeat as indicated Discard flow through 2x 35 mL 2 x 100 mL Elution Elute the plasmid DNA with Buffer N5 We recommend precipitating the eluate as soon as possible step 8 Nevertheless the eluate can be stored in closed vials on ice for several hours In this case the eluate should be preheated to room temperature before the plasmid DNA is precipitated 25 mL 100 mL Optional Determine plasmid yield by UV spectrophotometry in order to adjust the desired concentration of DNA step 10 28 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification Mega Giga AX 2000 AX 10000 8 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Mix carefully and centrifuge at 15 000 x g for 30 min at 4 C Carefully discard the supernatant 18 mL 70 mL 9 Wash and dry DNA pellet Add room temperature 70 ethanol to the pellet Vortex briefly an
27. e labeled with H and P phrases until 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze N2 N3 N5 Buffer salts ethanol Warning 226 210 233 5 20 403 235 Puffersalze Ethanol 5 20 Achtung 2 Sodium hydroxide Warning 315 319 280 302 352 0 5 2 0 305 351 338 Natriumacetat 0 5 2 0 Achtung 332 313 337 313 RNase A RNase A Iyophilized Danger 317 334 261 280 RNase A Iyophilisiert Gefahr 302 352 304 341 333 313 342 311 363 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 315 Causes skin irritation Verursacht Hautreizungen H 317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H 319 Causes serious eye irritation Verursacht schwere Augenreizung H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursa chen Precaution phrases P 210 Keep away from heat sparks open flames hot surfaces No smoking Von Hitze Funken offener Flamme heiBen Oberfl chen fernhalten Nicht rauchen P 233 Keep container tightly closed Beh lter dicht verschlossen halten P 261 Avoid breathing dust Einatmen von Staub vermeiden P 2
28. entations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract 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 general scientific information Applications mentioned in MACHEREY NAGEL literature are provided 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 correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 e mail tech bio mn net com Trademarks DH5a is a trademark of Life Technologies Inc NucleoBond is a trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services
29. f the procedure is causing the problem First the bacterial culture has to be checked for sufficient growth OD 0 in the presence of an appropriate selective antibiotic see Table 3 Second aliquots of the cleared lysate the flow through the combined washing steps Buffer N3 and the eluate should be kept for further analysis by agarose gel electrophoresis Table 3 Information about antibiotics according to Maniatis Antibiotic Stock solution Storage Working concentration concenitration Ampicillin 50 mg mL in H O 20 C 20 50 ug mL Carbenicillin 50 mg mL in H O 20 C 20 60 yg mL Chloramphenicol 34 mg mL in EtOH 20 C 25 170 pg mL Kanamycin 10 mg mL in H O 20 C 10 50 g mL Streptomycin 10 mg mL in H O 20 C 10 50 pg mL Tetracycline 5 mg mL in EtOH 20 C 10 50 g mL Maniatis T Fritsch EF Sambrook J Molecular cloning A laboratory manual Cold Spring Harbor Cold Spring New York 1982 36 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 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 binding capacity of each column size used for the preparation please also see Tables 1 and 2 Precipitate the nucleic acids by adding 0 7 volumes of isopropanol centrifuge the sample wash the pellet using 70 ethanol centrifuge again air dry for 10 minutes dissolve the DNA in 100 uL TE buffer pH 8 0 and
30. filter with a few drops of Buffer N2 and load the bacterial lysate onto the wet filter Either collect the flow through in a separate vessel and proceed with step 5 OR position funnel and filter directly on top of the NucleoBond Column to clear and load the lysate in one time saving step skip step 5 See section 3 5 for more information on how to set up the funnel and filter and for alternative clarification methods like centrifugation Binding Load the cleared lysate from step 4 onto the NucleoBond Column Allow the column to empty by gravity flow Optional You may want to save all or part of the flow through for analysis Washing Wash the column with Buffer N3 Repeat as indicated Discard flow through 2x 1 5 mL 10 mL Elution Elute the plasmid DNA with Buffer N5 MACHEREY NAGEL 11 2011 Rev 09 25 Plasmid DNA purification Mini Midi maxi AX 20 AX 100 AX 500 We recommend precipitating the eluate as soon as possible step 8 Nevertheless the eluate can be stored in closed vials on ice for several hours In this case the eluate should be preheated to room temperature before the plasmid DNA is precipitated Optional Determine plasmid yield by UV spectrophotometry in order to adjust the desired concentration of DNA step 10 8 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Mix carefully a
31. ion 2 1 Properties 2 2 About this user manual 3 Product description 3 1 The basic principle 3 2 Kit specifications 3 3 Buffer compositions 3 4 High low copy plasmid purification 3 5 Filtration of the lysate 3 6 Elution procedure 4 Storage conditions and preparation of working solutions 5 Safety instructions 5 1 Risk and safety phrases 5 2 GHS classification 6 Growing of bacterial cultures 6 1 General considerations 6 2 Selection of culture media 6 3 Difficult to lyse strains 6 4 Chloramphenicol amplification of low copy plasmids 7 NucleoBond plasmid purification 7 1 General procedure 7 2 High copy plasmid purification Mini Midi Maxi 7 3 High copy plasmid purification Mega Giga 7 4 Low copy plasmid purification Mini Midi 7 5 Low copy plasmid purification Maxi BAC Mega 11 11 12 13 13 13 14 15 16 17 18 19 19 20 22 22 23 23 23 24 24 24 27 30 33 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 8 Appendix 8 1 8 2 8 3 8 4 8 5 Determination of DNA yield and quality Troubleshooting Ordering information References Product use restriction warranty 36 36 36 43 44 45 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 1 Components 1 1 Kit contents NucleoBond PC 20 20 preps 100 preps REF 740571 740571 100 Resuspension Buffer S1 25 mL 2x25mL Lysis Buffer S2 25 mL 2x25mL Neutralization Buffer S3 25 mL 2x25mL Equilibr
32. ith a pipette tip Allow the pellet to dry at room temperature 18 25 C no less than the indicated time Drying for longer periods of time will not harm the quality of plasmid DNA but over drying may render the DNA less soluble 10 20 min 30 60 min 10 Reconstitute DNA Dissolve pellet in an appropriate volume of buffer TE or sterile deionized HO Depending on the type of centrifugation tube dissolve under 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 MACHEREY NAGEL 11 2011 Rev 09 35 Plasmid DNA purification 8 2 Appendix Determination of DNA yield and quality Plasmid yield is measured by UV spectroscopy by using the following relationship 1 OD at 260 nm 1 cm path length is equivalent to 50 ug plasmid DNA mL Plasmid quality is checked initially by running a 1 agarose gel This will give information on the percentage of ccc form structural integrity of isolated plasmid DNA Plasmid quality is checked by UV spectroscopy quotient Azg0 Aag0 A value of 1 80 1 90 is an indication for pure plasmid DNA Depending on further use of the purified plasmid more sophisticated analytical methods may have to be applied for quantification of byproducts Troubleshooting If you experience problems with reduced yield or purity it is recommended to check which purification step o
33. itive side effect of much less genomic DNA per plasmid but they obviously work only with plasmids that do not carry the chloramphenicol resistance gene Furthermore the method is only effective with low copy number plasmids under stringent control e g PBR322 All modern high copy number plasmids e g pUC are already under relaxed control due to mutations in the plasmid copy number control genes and show no significant additional increase in their copy number Maniatis T Fritsch EF Sambrook J Molecular cloning A laboratory manual Cold Spring Harbor Cold Spring New York 1982 Frenkel L Bremer H Increased amplification of plasmids pBR322 and pBR327 by low concentrations of chloramphenicol DNA 5 539 544 1986 MACHEREY NAGEL 11 2011 Rev 09 23 Plasmid DNA purification 7 7 1 NucleoBond plasmid purification General procedure Prepare an overnight culture Set up an overnight bacterial culture by inoculating the appropriate volume of LB medium plus antibiotic with a single colony picked from a freshly streaked plate Shake the culture overnight 12 16 h with selecting antibiotics added to the medium Centrifuge the culture at 6 000 x g for 15 min at 4 C Carefully discard the supernatant 7 2 High copy plasmid purification Mini Midi Maxi AX 20 AX 100 AX 500 1 Cultivate and harvest bacterial cells Harvest bacteria from an LB culture by centrifugation at 4 500 6 000 x
34. lic macro porous silica beads coupled to a methyl ethylamine functional group The functional group provides a high overall charge density that permits the negatively charged phosphate backbone of plasmid DNA to bind with high specificity to the resin Due to a specialized manufacturing process that is rigorously controlled and monitored the beads are uniform in diameter and contain particularly large pores These special properties allow for optimum flow rates through the column and more efficient binding of nucleic acids to the matrix Thus using the matrix you can achieve sharp well defined elution profiles for individual nucleic acid species see Figure 1 NucleoBond AX can separate distinct nucleic acids from each other and from proteins carbohydrates and other unwanted cellular components The purified nucleic acid products are suitable for use in the most demanding molecular biology applications including transfection in vitro transcription automated or manual sequencing cloning hybridization and PCR Plasmid DNA m large constructs _ Single stranded DNA Double stranded DNA mRNA 168 238 rRNA m 5S rRNA Compound class _ tRNA _ _ Proteins dyes polysaccharides metabolites trinucleotides rRNA Plasmid DNA large constructs Absorbance at 260 nm 0 0 5 1 1 5 Salt concentration for elution M KCl Figure 1 Elution profile of NucleoBond AX Resin at pH 7 0 The more in
35. mosomal DNA For Mini and Midi preps cultivation in flasks is recommended At least for Mega and Giga preps the use of an appropriate fermentation system is recommended in order to optimize cultivation conditions 6 3 Difficult to Iyse 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 S1 containing 2 mg mL lysozyme and incubate at 37 C for 30 minutes Proceed then with the lysis procedure according to the NucleoBond standard protocol 6 4 Chloramphenicol amplification of low copy plasmids To dramatically increase the low copy number of pMB1 colE1 derived plasmids grow the cell culture to mid or late log phase ODgoo 0 6 2 0 under selective conditions with an appropriate antibiotic Then add 170 ug mL chloramphenicol and continue incubation for a further 8 12 hours Chloramphenicol inhibits host protein synthesis and thus prevents replication of the host chromosome Plasmid replication however is independent of newly synthesized proteins and continues for several hours until up to 2000 3000 copies per cell are accumulated Alternatively the cell culture can be grown with only partial inhibition of protein synthesis under low chloramphenicol concentrations 10 20 pg mL resulting in a 5 10 fold greater yield of plasmid DNA Both methods show the pos
36. nd centrifuge at 15 000 x g for 30 min at 4 C Carefully discard the supernatant 0 75 mL 3 5 mL 11 0 mL 9 Wash and dry DNA pellet Add room temperature 70 ethanol to the pellet Vortex briefly and centrifuge at 15 000 x g for 10 min at room temperature 18 25 C 500 uL 2mL 5 mL Carefully remove ethanol from the tube with a pipette tip Allow the pellet to dry at room temperature 18 25 C no less than the indicated time Drying for longer periods of time will not harm the quality of plasmid DNA but over drying may render the DNA less soluble 5 10 min HETI 10 Reconstitute DNA Dissolve pellet in an appropriate volume of buffer TE or sterile deionized H O Depending on the type of centrifugation tube dissolve under 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 26 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 7 3 High copy plasmid purification Mega Giga Mega Giga AX 2000 AX 10000 1 Cultivate and harvest bacterial cells Harvest bacteria from an LB culture by centrifugation at 4 500 6 000 x g for 15 min at 4 C 2 Cell lysis Carefully resuspend the pellet of bacterial cells in Buffer S1 RNase A Please see section 6 3 regarding difficult to lyse strains 45 mL 120 mL Add Buffer S2 to the suspension Mix gentl
37. o dry at room temperature 18 25 C Drying for longer periods of time will not harm the quality of plasmid DNA but over drying may render the DNA less soluble 5 10 min 5 10 min Reconstitute DNA Dissolve pellet in an appropriate volume of buffer TE or sterile deionized HO Depending on the type of centrifugation tube dissolve under 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 32 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 7 5 Low copy plasmid purification Maxi BAC Mega Maxi Mega AX 500 BAC 100 AX 2000 1 Cultivate and harvest bacterial cells Harvest bacteria from an LB culture by centrifugation at 4 500 6 000 x g for 15 min at 4 C 2 Cell lysis Carefully resuspend the pellet of bacterial cells in Buffer S1 RNase A Please see section 6 3 regarding difficult to lyse strains 24 mL 90 mL Add Buffer S2 to the suspension Mix gently by inverting the tube 6 8 times Incubate the mixture at room temperature 18 25 C for 2 3 min max 5 min Do not vortex as this will release contaminating chromosomal DNA from cellular debris into the suspension 24 mL 90 mL Add pre cooled Buffer S3 4 C to the suspension Immediately mix the lysate gently by inverting the flask 6 8 time
38. oduct defects in products or MACHEREY NAGEL 11 2011 Rev 09 45 Plasmid DNA purification components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY 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 REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental 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 MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or repres
39. opy number plasmid is 1 3 mg per gram wet weight Table 2 Recommended culture volume Copy LB culture Wet weight Recommended Average yield plasmids volume of pellet column size High copy 1 5 mL AX 20 Mini 3 20 ug 5 30 mL AX 100 Midi 20 100 ug 30 150 mL 0 75g AX 500 Maxi 100 500 ug 150 500 mL 2 5 g AX 2000 Mega 500 ug 2 mg 500 2 000 mL 10g AX 10000 Giga 2 mg 10 mg Low copy 3 10 mL _ AX 20 Mini 3 20 ug 10 100 mL AX 100 Midi 20 100 ug 100 500 mL 1 5 2 2 g AX 500 Maxi 100 500 ug 100 500mL 1 5 2 29 BAC 100 Maxi 100 ug 500 2 000 mL 5 7 5 g AX 2000 Mega 500 ug 2 mg For AX 20 and AX 100 it is not necessary to measure the wet weight but depending on the media used ODgoo Should be determined For a low copy protocol using AX 10000 Giga columns please call our Technical Service Center 22 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 6 2 Selection of culture media The cultivation of cells is recommended at 37 C in LB Luria Bertani medium at con stant shaking 200 250 rpm Alternatively rich media like 2x YT Yeast Tryptone or TB Terrific Broth can be used By using 2xYT or TB bacteria grow faster and reach the stationary phase much earlier than in LB medium lt 2 h This may lead to a higher percentage of dead or starving cells when starting the preparation The resulting plasmid DNA from overgrown cultures may be partially degraded or contaminated with chro
40. orm well j in subsequent PNA is degraded reactions Make sure that all equipment pipettors centrifuge tubes etc are clean and nuclease free Make sure that the alkaline Iysis step i e the incubation of sample after addition of Buffer S2 does not proceed for longer than 5 min Culture volumes used are too large Reduce the culture volume or increase the amount of Buffer NucleoBond S1 S2 and S3 used during the lysis steps Folded Filters clog during ERN filtration Incubation time too short Make sure that S1 S2 S3 lysate was incubated according to the protocol 42 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 8 3 Ordering information Product REF Pack of NucleoBond PC 20 740571 20 preps 740571 100 100 preps NucleoBond AX 20 740511 20 columns NucleoBond PC 100 740573 20 preps 740573 100 100 preps NucleoBond AX 100 740521 20 columns 740521 100 100 columns NucleoBond PC 500 740574 10 preps 740574 25 25 preps 740574 50 50 preps 740574 100 100 preps NucleoBond AX 500 740531 10 columns 740531 50 50 columns NucleoBond PC 2000 740576 5 preps NucleoBond AX 2000 740525 10 columns NucleoBond PC 10000 740593 5 preps NucleoBond AX 10000 740534 5 columns NucleoBond Finalizer 740519 20 20 filters for use with NucleoBond PC 100 PC 500 2 syringe sets PC 500 EF NucleoBond Xtra Midi Midi EF NucleoBond Finalizer Plus 740520 20 20 filters for use with NucleoBond PC 100 PC 5
41. re reuse Kontaminierte Kleidung vor erneutem Waschen tragen Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort aufbewahren For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com MACHEREY NAGEL 11 2011 Rev 09 21 Plasmid DNA purification 6 Growing of bacterial cultures 6 1 General considerations Yield and quality of plasmid DNA depend on for example the type of growing media and antibiotics the bacterial host plasmid type size or copy number Therefore these factors should be taken into consideration For cultivation of bacterial cells we recommend LB medium The suggested bacterial culture volumes for each column size as well as expected plasmid yields are listed in Table 2 Overnight cultures in flasks usually reach under vigorous shaking an ODgo9 of 3 6 while fermentation cultures reach 10 and more Therefore please refer not only to the culture volume but also check ODgoo and pellet wet weight particularly if richer culture media like 2xYT or TB are used If too much bacterial material is used lysis and precipitation steps are inefficient and finally NucleoBond Columns are overloaded causing decreased yield and plasmid quality As a general rule 1 liter E coli culture grown in LB medium yields a pellet of about 3 20 g wet weight The expected yield for a high c
42. repeats or important handling steps are emphasized in bold type within the instruction Additional notes or optional steps are printed in italic In the example shown above the pellet of the bacterial cells has to be resuspended in 0 4 mL of Buffer S1 when performing a Mini prep using NucleoBond AX 20 Columns in 4 mL of Buffer S1 when performing a Midi prep using NucleoBond AX 100 Columns and in 12 mL of Buffer S1 when performing a Maxi prep using NucleoBond AX 500 Columns 12 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 3 3 1 Product description The basic principle NucleoBond PC BAC kits employ a modified alkaline SDS Iysis procedure to prepare the bacterial cell pellet for plasmid purification Both chromosomal and plasmid DNA are denatured under these alkaline conditions Potassium acetate is then added to the denatured lysate which causes the formation of a precipitate containing chromosomal DNA and other cellular compounds The potassium acetate buffer also neutralizes the lysate Plasmid DNA can revert to its native supercoiled structure and remains in solution After equilibrating the appropriate NucleoBond Column with equilibration buffer plasmid DNA is bound to the anion exchange resin and finally eluted after efficient washing of the column After precipitation of the eluted DNA it can easily be dissolved in TE buffer for further use 3 2 Kit specifications NucleoBond Plasmid purifi
43. s until a homogeneous suspension containing an off white flocculate is formed Incubate the suspension on ice for 5 min 24 mL 90 mL 3 Equilibration of the column Equilibrate a NucleoBond AX 500 Maxi BAC 100 Maxi or AX 2000 Mega Column with Buffer N2 Allow the column to empty by gravity flow Discard flow through 6 mL 20 mL 4 Clarification of the lysate Clear the bacterial lysate by following EITHER option 1 or option 2 described below This step is extremely important excess precipitate left in suspension may clog the NucleoBond Column in later steps Note For purification of BAC DNA it is recommended to follow option 1 MACHEREY NAGEL 11 2011 Rev 09 33 Plasmid DNA purification Maxi Mega AX 500 BAC 100 AX 2000 Note Complete removal of precipitated protein and cell debris is essential to avoid clogging of the NucleoBond Column Place a NucleoBond Folded Filter in a funnel of appropriate size Wet the filter with a few drops of Buffer N2 and load the bacterial lysate onto the wet filter Either collect the flow through in a separate vessel and proceed with step 5 OR position funnel and filter directly on top of the NucleoBond Column to clear and load the lysate in one time saving step skip step 5 See section 3 5 for more information on how to set up the funnel and filter and for alternative clarification methods like centrifugation
44. teractions a nucleic acid can form between the phosphate backbone and the positively 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 MACHEREY NAGEL 11 2011 Rev 09 11 Plasmid DNA purification 2 2 About this user manual It is strongly recommended reading the detailed protocol sections of this user manual if the NucleoBond Plasmid kit is used for the first time Experienced users however may refer to the Protocol at a glance instead The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure All technical literature is available on the internet at www mn net com The protocols in this manual are organized as follows The volumes of the respective buffers used for a particular column size are high lighted Each procedural step is arranged like the following example taken from section 7 2 High copy plasmid purification Mini Midi Maxi AX 20 AX 100 AX 500 1 Carefully resuspend the pellet of bacterial cells in Buffer S1 RNase A Please see section 6 3 regarding difficult to Iyse strains For example if you are performing a Mini prep to purify plasmid DNA you will find volumes or incubation times in the white boxes The name of the buffer buffer volume incubation times
45. top filter to a suitable flask e g Schott load the bacterial lysate and apply the vacuum see Figure 3 The lysate is cleared and sterile filtered within 3 5 minutes and can then be loaded onto the NucleoBond Column 3 Clearing the lysate by centrifugation is recommend ed for either very small sample volumes NucleoBond PC 20 or very large volumes low copy protocol Centrifuge the lysate for 5 30 minutes at gt 12 000 x g at room temperature or better 4 C Apply the cleared lysate directly to the NucleoBond Column or pass the lysate through a wet folded filter to remove remaining particles before loading the column Figure 3 Correct use of the NucleoBond Bottle Top Filter 16 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 3 6 Elution procedure Elution is carried out into a new tube with the volume of elution buffer indicated in the corresponding protocol The plasmid DNA is precipitated by the addition of room temperature 18 25 C isopropanol Do not let the plasmid DNA solution drop into a vial with isopropanol because this leads to spontaneous co precipitation of salt Only use room temperature 18 25 C isopropanol to prevent spontaneous co precipitation of salt MACHEREY NAGEL 11 2011 Rev 09 17 Plasmid DNA purification 4 Storage conditions and preparation of working solutions Attention Buffer S2 contains sodium dodecylsulfate and sodium hydroxide Wear gloves an
46. uch as Triton X 100 and NP 40 3 3 Buffer compositions Resuspension Buffer S1 50 mM Tris HCl 10 mM EDTA 100 ug mL RNase A pH 8 0 Lysis Buffer S2 200 mM NaOH 1 SDS Neutralization Buffer S3 2 8 M KAc pH 5 1 Equilibration Buffer N2 100 mM Tris 15 ethanol 900 mM KCI 0 15 Triton X 100 adjusted to pH 6 3 with HPO Wash Buffer N3 100 mM Tris 15 ethanol 1 15 M KCI adjusted to pH 6 3 with HPO Elution Buffer N5 100 mM Tris 15 ethanol 1 M KCI adjusted to pH 8 5 with HPO Note Keep all buffers tightly capped The concentration of KCI required for eluting the desired nucleic acid is highly dependent on the pH value of the eluent For this reason pH values must be carefully controlled if the buffers have been prepared by the customer A deviation of more than 0 1 pH unit from the given values may affect yields If you are consistently experiencing reduced yields check the pH of all buffers before continuing Buffers should be adjusted with HPO or KOH 14 MACHEREY NAGEL 11 2011 Rev 09 Plasmid DNA purification 3 4 High low copy plasmid purification NucleoBond PC kits are recommended for the isolation of high copy plasmids gt 20 copies cell however low copy plasmids lt 20 copies cell can be isolated as well If you are purifying low copy plasmids you will need to supplement the NucleoBond PC kits with additional buffers We recommend the NucleoBond Buffer Set REF 740601 for
47. y by inverting the tube 6 8 times Incubate the mixture at room temperature 18 25 C for 2 3 min max 5 min Do not vortex as this will release contaminating chromosomal DNA from cellular debris into the suspension 45 mL 120 mL Add pre cooled Buffer S3 4 C to the suspension Immediately mix the lysate gently by inverting the flask 6 8 times until a homogeneous suspension containing an off white flocculate is formed Incubate the suspension on ice for 5 min 45 mL 120 mL 3 Equilibration of the column Equilibrate a NucleoBond AX 2000 Mega AX 10000 Giga Column with Buffer N2 Allow the column to empty by gravity flow Discard flow through 20 mL 100 mL MACHEREY NAGEL 11 2011 Rev 09 27 Plasmid DNA purification Mega Giga AX 2000 AX 10000 Clarification of the Iysate Note Complete removal of precipitated protein and cell debris is essential to avoid clogging of the NucleoBond Column For NucleoBond PC 2000 place a NucleoBond Folded Filter XL in a funnel of appropriate size For NucleoBond PC 10000 put a NucleoBond Folded Filter of type 1 into a folded filter of type 2 and place the combination in a funnel Wet the filter s with a few drops of Buffer N2 and load the bacterial lysate onto the wet filter s Either collect the flow through in a separate vessel and proceed with step 5 OR position funnel and filter directly on top of th
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