NucleoSpin® 96 Plasmid (Core Kit) - MACHEREY
Contents
1. Plasmid DNA purification User manual NucleoSpin 96 Plasmid NucleoSpin 96 Plasmid Core Kit April 2014 Rev 03 MACHEREY NAGEL MN www mn net com Plasmid DNA purification Table of contents 1 Components 1 1 Kit contents 1 2 Reagents to be supplied by user 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 6 2 3 Required hardware 7 2 4 Recommended accessories for use of the NucleoSpin 96 Plasmid Core Kit 8 2 5 Automated processing on robotic platforms 9 2 6 Growth of bacterial cultures 10 2 6 1 Selection of culture media 10 2 6 2 Cultivation of bacteria in a Square well Block 10 2 6 3 Cultivation of bacteria in tubes 10 2 7 Elution procedures 11 3 Storage conditions and preparation of working solutions 12 4 Safety instructions 13 5 Protocols 15 5 1 NucleoSpin 96 Plasmid manual vacuum processing 15 5 2 NucleoSpin 96 Plasmid elution of DNA using a centrifuge 23 6 Appendix 24 6 1 Troubleshooting 24 6 2 Ordering information 27 6 3 Product use restriction warranty 28 MACHEREY NAGEL 04 2014 Rev 03 3 Plasmid DNA purification 1 Components 1 1 Kit contents NucleoSpin 96 Plasmid 1x 96 preps 4x96 preps 24x 96 preps REF 740625 1 740625 4 740625 24 Resuspension Buffer A1 75 mL 150 mL 6x 150 mL Lysis Buffer A2 75 mL 150 mL 6x 150 mL Neutralization Buffer A3 100 mL 200 mL 6 x 200 mL Wash Buffer AW 100 mL 400 mL 6 x 400 mL Wash B2. the NucleoSpin Plasmid Binding Plate slightly and collect the eluted DNA After the elution buffer has passed the wells release vacuum Remove the Elution Plate U bottom containing eluted DNA and seal the strips plate with adhesive cover foil or Cap Strips respectively for further storage Reduction of atmospheric pressure 22 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 96 Plasmid elution of DNA using a centrifuge 5 2 NucleoSpin 96 Plasmid elution of DNA using a centrifuge Elution of purified DNA in a centrifuge can be performed be necessary when higher concentrations of the final DNA are required for downstream applications Using a centrifuge allows the dispensed volume to be reduced down to 50 75 uL Required hardware For centrifugation a microtiterplate centrifuge that can accommodate the NucleoSpin Plasmid Binding Plate stacked on a rack of Tube Strips is required bucket height 85 mm It is also necessary that the centrifuge reaches accelerations of 5 600 6 000 x g Suitable elution tubes Rack of Tube Strips have to be ordered separately see ordering information 1 Stop the method after the final washing step with Buffer A4 Remove the NucleoSpin Plasmid Binding Plate from the manifold s top and tap on a sheet of filter paper to remove residual wash buffer from the outlets 2 Place the NucleoSpin Plasmid Binding Plate on top of a MN Square well Block not included in the ki
3. 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 0 24 21 969 270 e mail tech bio mn net com Trademarks Disclaimer NucleoSpin is a registered 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 we can not grant any guarantees regarding selection efficiency or operation 30 MACHEREY NAGEL 04 2014 Rev 03
4. Rev 03 5 Plasmid DNA purification 2 Product description 2 1 The basic principle The NucleoSpin 96 Plasmid procedure is a modified version of the Birnboim and Doly alkaline Iysis plasmid Mini prep protocol Bacterial cultures are harvested by an initial centrifugation step After resuspension of the pelleted bacteria Buffer A1 and alkaline cell lysis Buffer A2 a neutralization and binding buffer Buffer A3 containing chaotropic salts is added Resulting bacterial crude lysates are cleared by vacuum filtration with the NucleoSpin Plasmid Filter Plate The cleared lysates containing the plasmid DNA are collected into the NucleoSpin Plasmid Binding Plate The chaotropic salt leads to a reversible adsorption of the plasmid DNA to the NucleoSpin silica membrane during the second vacuum filtration step High purity of the final plasmid DNA preparation is achieved by complete removal of cellular contaminants salts detergents and other compounds by subsequent washing steps Highly pure plasmid DNA is finally eluted with Elution Buffer AE 5 mM Tris HCl pH 8 5 or water pH 8 0 8 5 and can directly be used for downstream applications 2 2 Kit specifications NucleoSpin 96 Plasmid is designed for the manual or automated large scale purification of high copy plasmid DNA from E coliin the 96 well plate format NucleoSpin 96 Plasmid kits REF 740625 1 4 24 are supplied with all accessory plates for highest convenience
5. The NucleoSpin 96 Plasmid Core Kit REF 740616 4 24 provides the buffers RNase A NucleoSpin Plasmid Filter Plates and NucleoSpin Plasmid Binding Plates Accessory components e g culture plate elution plate MN Wash Plate and Wash Buffer AW are not provided with the core kit but can be individually selected from a variety of suitable accessories see section 2 4 for further information This allows highest flexibility for the user Please note All given specifications or information in this manual refer equally to the NucleoSpin 96 Plasmid kit REF 740625 1 4 24 as well as to the NucleoSpin 96 Plasmid Core Kit REF 740616 4 24 The kits allow for easy automation on common liquid handling instruments For more information about the automation process and the availability of ready to run scripts for certain platforms please refer to section 2 5 and or contact your local distributor or MN directly Using the NucleoSpin 96 Plasmid kits allow simultaneous manual processing of up to 96 samples typically within less than 45 minutes Actual processing time depends on the configuration of the liquid handling system used 1 Birnboim H C amp Doly J 1979 Nucleic Acids Res 7 1513 1523 6 MACHEREY NAGEL 04 2014 Rev 03 Plasmid DNA purification Typically yields of 5 15 ug plasmid DNA can be purified from 1 5 mL overnight cultures Yield depends on copy number and plasmid size selected culture medium a
6. Buffer AW RNase A and NucleoSpin Filter Binding Plates Accessory plates e g culture blocks elution plates are not provided with the core kit The user can individually select additional consumables from a variety of suitable accessory plates according to his requirements for highest flexibility For use of NucleoSpin 96 Plasmid Core Kit follow the standard protocols see section 5 1 or 5 2 respectively Recommended accessories for use of the NucleoSpin 96 Plasmid Core Kit are available from MACHEREY NAGEL For ordering information please refer to section 6 2 Protocol Suitable consumables not Remarks step supplied with the core kits Cultivation of Culture Plates bacteria Square well Blocks with Gas permeable Foil Wash step MN Wash Plates MN Wash Plate minimizes the risk of cross contamination vacuum processing only Buffer AW Recommended additional wash buffer for bacterial host strain with high endogenous nuclease activity e g E coli HB 101 BMH 71 18 mutS JM or any wildtype strains or for improvement of sequencing results Elution Elution Plate U bottom or Rack of Tube Strips including Cap Strips Not suitable for elution by centrifugation 8 MACHEREY NAGEL 04 2014 Rev 03 Plasmid DNA purification 2 5 Automated processing on robotic platforms NucleoSpin 96 Plasmid can be used fully automated on many common laboratory worksta
7. KT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter sptilen Rinse mouth Mund 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 oder ausschlag 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 doctor Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM Arzt anrufen Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen 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 14 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 96 Plasmid manual vacuum processing 5 Protocols 5 1 NucleoSpin 96 Plasmid manual vacuum processing For hardware requirements refer to section 2 3 For detailed information regarding the vacuum manifold setup see page 18 19 For detailed information on each step see page 20 For use of the NucleoSpin 96 Plasmid Co
8. ar 1 min If necessary press down the NucleoSpin Plasmid Binding Plate slightly Allow the buffer to pass the wells Release the vacuum Note This additional wash step is recommended if the bacterial host strain has a high endogenous nuclease activity e g E coli HB 101 BMH 71 18 mutS JM or any wild type strains or if sequencing results need to be improved Add 900 uL Buffer A4 with ethanol to each well Apply vacuum of 0 2 to 0 4 bar 1 min and allow the buffer to pass the wells Release the vacuum Repeat the wash step with 900 uL Buffer A4 Apply vacuum of 0 2 to 0 4 bar 1 min and allow the buffer to pass the wells Release the vacuum 10 Remove MN Wash Plate After the final washing step close the valve release the vacuum and remove the NucleoSpin Plasmid Binding Plate Remove manifold lid MN Wash Plate and waste container from the vacuum manifold Reduction of atmospheric pressure MACHEREY NAGEL 04 2014 Rev 03 21 NucleoSpin 96 Plasmid manual vacuum processing 11 Dry NucleoSpin Plasmid Binding Plate Remove any residual wash buffer from the NucleoSpin Plasmid Binding Plate If necessary tap the outlets of the plate onto a clean paper sheet supplied with the MN Wash Plate or soft tissue until no drops come out Close the manifold base with the manifold lid Place the NucleoSpin Binding Plate on top of the manifold Apply vacuum of 0 4 to 0 6 bar f
9. asmid DNA with TE buffer EDTA may inhibit enzymatic reactions like DNA sequencing Repurify the plasmid DNA and elute with Buffer AE or nuclease free water Alternatively the plasmid DNA may be precipitated with ethanol and redissolved in Buffer AE or nuclease free water E coli strains with high endogenous nuclease levels are used as host Perform the washing step with Buffer AW before washing with ethanolic Buffer A4 Not enough DNA used for sequencing reactions Quantitate DNA by agarose gel electrophoresis before setting up sequencing reactions 26 MACHEREY NAGEL 04 2014 Rev 03 Plasmid DNA purification 6 2 Ordering information Product REF Pack of NucleoSpin 96 Plasmid 740625 1 1x 96 preps 740625 4 4x 96 preps 740625 24 24 x 96 preps NucleoSpin 96 Plasmid Core Kit 740616 4 4 x 96 preps 740616 24 24 x 96 preps NucleoSpin 8 Plasmid 740621 12 x 8 preps 740621 5 60 x 8 preps NucleoSpin 8 Plasmid Core Kit 740461 4 48 x 8 preps Buffer A1 740911 1 1L without RNase A Buffer A2 740912 1 1L Buffer A3 740913 1 1L Buffer A4 Concentrate 740914 1 200 mL for 1 L Buffer A4 Buffer AW 740916 1 1L Buffer AE 740917 1 1L RNase A lyophilized 740505 100 mg NucleoVac 96 Vacuum Manifold 740681 1 NucleoVac Vacuum Regulator 740641 1 Round well Block with Cap Strips 740475 4 sets 740475 24 24 sets Rack of Tube Strips 740477 4 sets 1 set consists of 1 rack 12 strips 740477 24 24 sets with 8 tu
10. bes each and 12 Cap Strips Cap Strips 740478 48 740478 24 288 MN Square well Block 740476 4 740476 24 24 MACHEREY NAGEL 04 2014 Rev 03 Plasmid DNA purification Product REF Pack of MN Wash Plate 740479 4 740479 24 24 Culture Plate 740488 4 sets with Gas permeable Foil 740488 24 24 sets Elution Plate U bottom 740486 24 24 sets with Self adhering Foil Gas permeable Foil 740675 50 Self adhering Foil 740676 50 Visit www mn net com for more detailed product information 6 3 Product use restriction warranty NucleoSpin 96 Plasmid Core 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 applica
11. e 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 hot surfaces sparks open flames and other ignition sources No smoking Von Hitze hei en Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen MACHEREY NAGEL 04 2014 Rev 03 13 Plasmid DNA purification P 233 P 261 P 280 P 301 312 P 302 352 P 304 340 P 305 351 338 P 330 P 332 313 P 333 313 P 337 313 P 342 311 P 363 P 403 235 Keep container tightly closed Beh lter dicht verschlossen halten Avoid breathing dust Einatmen von Staub vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF SWALLOWED Call a POISON CENTER doctor if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM Arzt anrufen IF ON SKIN Wash with plenty of water Bei Kontakt mit der Haut Mit viel Wasser waschen IF INHALED Remove victim to fresh air and keep at rest in a position com fortable for breathing Bei Einatmen 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 KONTA
12. ee the following table for correlation between the dispensed elution buffer volume and typical recoveries following the standard protocol under vacuum The recommended dispense volume of elution buffer is 125 uL Correlation between dispensed elution buffer volume and typical recovery Dispensed elution buffer 75 uL 100 uL 125 uL 150 uL 175 uL Recovered elution buffer containing PCR products 30 5 uL 55 5 ul 80 5 jL 105 5 jL 1130 5 uL Recovered DNA ug Concentration ng pL a Recovery 250 100 90 200 l o 2 80 amp 2 lt 5 150 3 S 6 a 8 5 3 mee ot v 3 3 2 100 g 8 a 60 50 50 60 80 100 120 140 160 180 200 Dispensed elution buffer pL mee 20 40 60 80 100 120 140 160 Recovered elution buffer pL Figure 1 Recovery rate and concentration depend on elution volume 10 ug of pBluescript pasmid were purified with NucleoSpin 96 Plasmid and eluted with the indicated elution buffer volumes MACHEREY NAGEL 04 2014 Rev 03 11 Plasmid DNA purification 3 Storage conditions and preparation of working solutions Attention Buffers A3 and AW contain chaotropic salts which are irritant Buffer A2 contains SDS and sodium hydroxide which are irritant and hazardous Wear gloves and goggles CAUTION Buffers A3 and AW contain guanidine hydrochloride which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solution
13. erature MACHEREY NAGEL 04 2014 Rev 03 9 Plasmid DNA purification 2 6 Growth of bacterial cultures 2 6 1 Selection of culture media The cultivation of cells is recommended at 37 C in LB Luria Bertani medium at constant shaking 200 250 rpm Alternatively rich media like 2x YT or TB Terrific Broth can be used By using 2x YT or TB bacteria grow faster and reach the stationary phase much earlier than in LB medium lt 12 h in culture tubes or flasks 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 chromosomal DNA 2 6 2 Cultivation of bacteria in a Square well Block Use the 96 well Square well Block Culture Plate not included in the core kits for growing bacteria Add 1 2 1 5 mL of selected medium with appropriate antibiotic e g 100 pg mL ampicillin to each well of the Square well Block To avoid cross contamination due to spillage during incubation do not exceed a total culture volume of 1 5 mL Inoculate each well with a single bacterial colony Cover the Square well Block with the Gas permeable Foil Grow the culture in a suitable incubator at 37 C for 16 24 h with vigorous shaking 200 400 rpm The Square well Block may be fixed to the shaker with large size flask clamps for 2 L flasks or tape Note The yield of plasmid DNA depends on growth conditions bacteria
14. etely into the wells of the NucleoSpin Plasmid Filter Plate Note Mix the suspension by pipetting up and down the entire volume once before transfer to the NucleoSpin Plasmid Filter Plate 6 Clear crude lysate by vacuum filtration Apply vacuum of 0 2 to 0 4 bar 1 5 min If necessary press down the NucleoSpin Plasmid Filter Plate slightly until flow through starts Adjust vacuum to establish a flow rate of 1 2 drops per second When the crude lysate has passed the NucleoSpin Plasmid Filter Plate release the vacuum 7 Reassemble vacuum manifold Remove and discard the NucleoSpin Plasmid Filter Plate Open the manifold lid Remove the NucleoSpin Plasmid Binding Plate white rings with cleared lysates Insert the MN Wash Plate on the spacers inside the manifold base see page 19 Close the manifold base with the manifold lid Place the Binding Plate on top of the manifold Reduction of atmospheric pressure 20 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 96 Plasmid manual vacuum processing 8 Bind DNA to silica membrane Apply vacuum of 0 2 to 0 4 bar 1 min If necessary press down the NucleoSpin Plasmid Binding Plate slightly until flow through starts Adjust vacuum to establish a flow rate of 1 2 drops per second When the cleared lysate has drained off release the vacuum 9 Wash silica membrane 1 wash optional Add 600 uL Buffer AW to each well Apply vacuum of 0 2 to 0 4 b
15. f the bottle Incubate ec n Buffer A2 at 30 40 C for 5 min and mix well before use Too many bacterial cells used Usage of LB as the growth medium is recommended When using rich media like TB cultures reach very high cell densities Reduce culture volume to 1 0 1 5 mL No or not enough antibiotic used during cultivation Cells harboring the plasmid of interest may become overgrown by non transformed cells Add appropriate amounts of freshly prepared stock solutions of antibiotic to all media Overgrown bacterial cultures See suggestions in section 2 6 Growth of bacterial cultures Poor er High copy number plasmid was not used l er Use high copy number plasmid Incomplete Iysis of bacterial cells See Possible cause and suggestions above No ethanol was added to Buffer A4 Concentrate ethanol evapo rated Add indicated volume of ethanol to Buffer A4 Concentrate and mix Keep bottle tightly closed to prevent evaporation of ethanol 24 MACHEREY NAGEL 04 2014 Rev 03 Plasmid DNA purification Problem Possible cause and suggestions Poor Elution conditions are not optimal plasmid If possible use a slightly alkaline elution buffer like Buffer AE yield 5 mM Tris HCl pH 8 5 When using nuclease free water for continued elution make sure the pH value is within the range of pH 8 0 8 5 Elution efficiencies drop drastically at pH lt 7 Excessive mixing steps Reduce number of mixi
16. in Plasmid Binding Plate Full vacuum by applying vacuum 10 15 min H Optional Dry the outlets of the NucleoSpin run pump continuously Plasmid Binding Plate by placing it on a sheet of filter paper before applying vacuum 12 Insert Elution Plate U bottom 13 Elute plasmid DNA 75 150 uL AE Optional Incubate 1 3 min 0 4 to 0 6 bar 1 min Reduction of atmospheric pressure 16 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 96 Plasmid manual vacuum processing Setup of vacuum manifold Lysate clearing Lysate clearing Step 4 Place the NucleoSpin Filter Plate on top of the manifold Step 3 Place the manifold lid on top of the manifold base Step 2 Place the NucleoSpin Binding Plate into the manifold Step 1 Insert spacers MTP Multi 96 Plate in the manifold base Final setup MACHEREY NAGEL 04 2014 Rev 03 17 NucleoSpin 96 Plasmid manual vacuum processing Setup of vacuum manifold Binding Washing Elution steps Binding Washing steps Elution step Step 4 Place the NucleoSpin Binding Plate on top of the manifold lid Step 3 Place the manifold lid on top of the manifold base Step 2 Place the MN Wash Plate in the manifold Step 1 Insert spacers MTP MULTI 96 PLATE in the manifold base Final setup Step 4 Place the NucleoSpin Binding Plate on top of the manifold lid Step 3 Place the manif
17. l strain and cell density of the culture as well as on the size and copy number of the vector Use of high copy number plasmids such as pUC pBluescript or pGEM and E coli strains like DH5a or XL1 Blue are recommended Growth times of 16 24 h are usually sufficient However for poorly growing bacteria prolonged incubation times of up to 30 h may be required 2 6 3 Cultivation of bacteria in tubes Use 1 5 mL of appropriate culture medium Depending on the bacterial strain and copy number of the plasmid up to 5 mL LB medium or 2 5 mL 2x YT or 2 5 mL TB medium can be used Grow bacteria with vigorous shaking 200 250 rpm for 10 14 h Optional If the liquid handling instrument does not allow for the use of selected culture tubes transfer the bacterial culture from the tubes into a suitable Square well Block For this transfer 1 5 mL of the culture to each well of the Square well Block Harvest the cultures by centrifugation Discard supernatant Usually 1 5 mL of culture are sufficient for DNA preparation However if necessary add additional 1 0 1 5 mL bacterial culture to each well of the Square well Block centrifuge again and discard the supernatant Do not use more than 5 mL LB culture or 2 5 mL rapid growing bacterial strain using 2x YT or TB medium because lysis efficiency might be lower when using cell pellets which are too large 10 MACHEREY NAGEL 04 2014 Rev 03 Plasmid DNA purification 2 7 Elution procedures S
18. nd bacterial host strain The DNA binding capacity is about 20 ug The final concentration of the eluted DNA is 50 200 ng uL depending on the elution buffer volume and the bacterial culture Typically the Aggo Azgo ratio is gt 1 8 Eluted DNA is ready to use for many downstream applications Kit specifications at a glance Parameter NucleoSpin 96 Plasmid Format 96 well plates Processing Manual or automated vacuum Lysate clarification 96 well filter plates Sample material 1 5 mL E coli culture Vector size lt 15 kbp Typical yield 4 6 ug mL E coli culture Elution volume 75 150 uL Preparation time 45 min plate Binding capacity 20 ug 2 3 Required hardware This kit is intended for use under vacuum A support protocol for elution under centrifugation is included see section 5 2 A support protocol for complete processing under centrifugation is available from our technical service tech bio mn net com The NucleoSpin 96 Plasmid kits can be used manually with the NucleoVac 96 Vacuum Manifold see ordering information Additionally a suitable centrifuge for harvesting the bacteria either plate or tube centrifuge and for the optimal elution step under centrifugation is required MACHEREY NAGEL 04 2014 Rev 03 Plasmid DNA purification 2 4 Recommended accessories for use of the NucleoSpin 96 Plasmid Core Kit The NucleoSpin 96 Plasmid Core Kit provides buffers except optional Wash
19. ng cycles reduce shaker action after addition of Lysis Buffer A2 and Neutralization Buffer A3 or before transfer of crude lysate to the NucleoSpin Plasmid Filter Strips Mixing will cause shearing of chromosomal DNA leading to a co purification during the preparation of plasmid DNA Culture volume was too high Reduce culture volume if lysate is too viscous for gentle and complete mixing Contamina tion with Bacterial culture overgrown chromosom al DNA Overgrown bacterial cultures contain lysed cells and degraded DNA See suggestions in section 2 6 Growth of bacterial cultures Lysis was too long e Lysis step must not exceed 5 min Tips Use wide bore disposable tips for transfer of crude lysate to the NucleoSpin Plasmid Filter Plate to prevent shearing of chromosomal DNA RNA was not degraded completely te mthe Ensure that RNase A was added to Buffer A1 before use eluate Reduce culture volume if necessary MACHEREY NAGEL 04 2014 Rev 03 25 Plasmid DNA purification Problem Possible cause and suggestions Suboptimal performance of plasmid DNA in sequencing reactions problems with downstream applications Carry over of ethanol inhibition of downstream analysis or problems with sample loading onto agarose e Be sure to remove all of ethanolic Buffer A4 after the final washing step Dry the NucleoSpin Plasmid Binding Plate for at least 10 min with maximum vacuum Elution of pl
20. o each bottle to each bottle 12 MACHEREY NAGEL 04 2014 Rev 03 Plasmid DNA purification 4 Safety instructions GHS classification Only harmful features do not need to be labeled with H and P phrases up to 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 A2 Sodium hydroxide Warning 315 319 280 302 352 0 5 2 0 305 351 338 Natriumhydroxid L sung Achtung 332 313 0 5 2 0 337 313 A3 Guanidine hydrochloride Warning 302 319 280 301 312 36 50 305 351 338 Guanidinhydrochlorid 36 50 Achtung 330 337 313 AW Guanidine hydrochloride Warning 226 302 210 233 280 36 50 isopropanol 319 301 312 20 50 305 351 338 Guanidinhydrochlorid 36 50 Achtung 330 337 313 Isopropanol 20 50 403 235 RNase A RNase A lyophilized Danger 317 334 261 280 RNase A lyophilisiert D Gefahr 302 352 304 340 333 313 342 311 363 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken 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 schwer
21. old lid on top of the manifold base Step 2 Place the Elution Plate in the manifold Step 1 Insert spacers MTP MULTI 96 PLATE in the manifold base Final setup 18 MACHEREY NAGEL 04 2014 Rev 03 NucleoSpin 96 Plasmid manual vacuum processing Detailed protocol For processing of NucleoSpin 96 Plasmid under vacuum the NucleoVac 96 Vacuum Manifold is required Before starting the preparation Check if Buffer A1 and Buffer A4 were prepared according to section 3 1 Cultivate and harvest bacterial cells Centrifuge the bacteria cultures 1 5 5 mL LB or up to 2 5 mL 2 x YT or TB for 10 min at 1 000 x g It is highly recommended centrifuging the bacterial cultures under the above mentioned conditions Centrifugation at higher g forces might produce tight pellets which are more difficult to resuspend Discard supernatant Remove residual medium by tapping tube or plate upside down on a clean paper sheet or soft tissue Optional Transfer bacteria cultures grown in tubes to a Square well Block Alternatively perform the next three steps in the tubes 2 Resuspend bacterial cells Add 250 uL Buffer A1 with RNase A to each sample Resuspend the bacterial pellet by vortexing or mixing by pipetting up and down Resuspend bacterial cells completely No clumps should be visible 3 Lyse bacterial cells Add 250 uL Buffer A2 to the suspension For lysis in tubes close the culture tube and mi
22. or at least 10 15 min to dry the membrane completely Run vacuum pump continuously Typically the adjusted vacuum is not reached at this step Achieving and keeping a continuous air flow in order to evaporate the remaining ethanol from Wash Buffer A4 is of more importance than reaching the precise mentioned atmospheric pressure Note The ethanol in Buffer A4 inhibits enzymatic reactions and has to be completely removed before eluting the DNA Finally release the vacuum 12 Insert Elution Plate U bottom Remove the manifold lid with the NucleoSpin Plasmid Binding Plate from the vacuum manifold Insert the Elution Plate on the spacers inside the manifold base Close the manifold base with the manifold lid Place the NucleoSpin Plasmid Binding Plate white rings on top of the manifold For elution into microtiter plates spacers MTP Multi 96 Plate are required which are already inserted into the manifold base from the previous steps 13 Elute plasmid DNA Elute the DNA by adding 125 uL Buffer AE 5 mM Tris HCl pH 8 5 125 uL is recommended a volume range of 75 150 uL is possible see section 2 7 or sterile distilled water pH 7 5 8 5 to each well of the NucleoSpin Plasmid Binding Plate The elution buffer should be dispensed carefully onto the center of the silica membrane Incubate the buffer on the membrane for 1 3 minutes at room temperature Apply vacuum of 0 4 to 0 6 bar 1 min If necessary press down
23. 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 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 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 04 2014 Rev 03 29 Plasmid DNA purification MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Last updated 07 2010 Rev
24. re Kit REF 740616 4 24 refer to section 2 4 regarding recommended accessories Before starting the preparation Check if Buffer A1 and Buffer A4 were prepared according to section 3 Set up the vacuum according to the sheme Protocol at a glance 1 Cultivate and harvest bacterial cells 1 5 mL 5 mL LB or up to 2 5 mL 2x YT or TB 10 min 1 000 x g 2 Resuspend bacterial cells 250 uL A1 Mix or shake 3 Lyse bacterial cells 250 uL A2 RT 2 5 min Shake 4 Neutralize 350 uL A3 Mix or shake Prepare vacuum manifold for lysate clearing step 5 Transfer crude lysates to NucleoSpin Plasmid Filter Plate violet rings MACHEREY NAGEL 04 2014 Rev 03 15 NucleoSpin 96 Plasmid manual vacuum processing 6 Clear crude lysates by vacuum filtration 0 2 to 0 4 bar directly into the NucleoSpin Plasmid 1 5 min Binding Plate white rings Optional Incubate 1 3 min before applying vacuum 7 Reassemble vacuum manifold Discard NucleoSpin Plasmid Filter Plate Remove NucleoSpin Plasmid Binding Plate with cleared lysates and insert MN Wash Plate Place NucleoSpin Plasmid Binding Plate on top of the manifold 8 Bind DNA to silica membrane of the 0 2 to 0 4 bar NucleoSpin Plasmid Binding Plate by 1 min applying vacuum 9 Wash silica membrane Optional 600 uL AW 900 uL A4 900 uL A4 0 2 to 0 4 bar 1 min each step 10 Remove MN Wash Plate 11 Dry NucleoSp
25. s directly to the sample preparation waste Storage conditions All kit components can be stored at room temperature 18 25 C and are stable up to one year Always keep buffer bottles tightly closed especially if buffers are preheated during the preparation Sodium dodecyl sulfate SDS in Buffer A2 may precipitate if stored at temperatures below 20 C If a precipitate is observed in Buffer A2 incubate the bottle at 30 40 C for several minutes and mix well Before starting any NucleoSpin 8 Plasmid protocol prepare the following Before the first use of the kit add 1 mL of Buffer A1 to the RNase A vial and vortex Transfer all of the resulting solution into the Buffer A1 bottle and mix thoroughly Indicate date of RNase A addition Store Buffer A1 containing RNase A at 4 C The solution will be stable at this temperature for at least six months Wash Buffer A4 Add the indicated volume of ethanol 96 100 to Buffer A4 Concentrate before use Mark the label of the bottle to indicate that ethanol was added NucleoSpin 96 Plasmid 1x 96 preps 4x 96 preps 24 x 96 preps REF 740625 1 740625 4 740625 24 Wash Buffer A4 100 mL 200 mL 6x 200 mL Concentrate Add 400 mL ethanol Add 800 mL ethanol Add 800 mL ethanol to each bottle to each bottle NucleoSpin 96 Plasmid Core Kit 4 x 96 preps 24 x 96 preps REF 740616 4 740616 24 Wash Buffer A4 2x 100 mL 12x 100 mL Concentrate Add 400 mL ethanol Add 400 mL ethanol t
26. tion 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 VITRO diagnostic products are expressiy 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 DIAGNOSTIC 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 28 MACHEREY NAGEL 04 2014 Rev 03 Plasmid DNA purification 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
27. tions For the availability of scripts and general considerations about adapting NucleoSpin 96 Plasmid on a certain workstation please contact MN Full processing under vacuum enables complete automation without the need of centrifugation steps regarding the drying of the binding membrane and elution step The risk of cross contamination is reduced by optimized vacuum settings during the elution step and by the improved shape of the outlets of the NucleoSpin Plasmid Binding Plate Drying of the NucleoSpin Plasmid Binding Plate under vacuum is sufficient because the bottom of the plate is protected from residues of wash buffer during the washing steps by the MN Wash Plate As a result we recommend trying to integrate the MN Wash Plate into the automated procedure The MN Frame see ordering information can be used to position the disposable MN Wash Plate inside the vacuum chamber This also reduces the risk of cross contamination as common metal adaptors tend to get contaminated by gDNA Thorough cleaning of the vacuum chamber is recommended after each run to prevent forming of gDNA containing aerosols Visit MN online at www mn net com or contact your local MACHEREY NAGEL distributor for technical support regarding hardware software setup instructions and selection of the protocol Several application notes of the NucleoSpin 96 Plasmid kit on various liquid handling instruments can also be found at www mn net com under Bioanalysis Lit
28. 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 product defects in products or 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
29. ts see ordering information and centrifuge for 10 min at maximum speed gt 4 000 x g optimal 5 800 x g Note Do not use a microtiter plate as a support for the NucleoSpin Plasmid Binding Plate Microtiter plates may crack under centrifugation at gt 1 500 x g 3 Place the NucleoSpin Plasmid Binding Plate on top of a Rack of Tube Strips Dispense Elution Buffer AE 50 150 uL directly onto the silica membrane and incubate for 1 3 min at room temperature Note Do not use a microtiter plate as elution plate Microtiter plates may crack under centrifugation at gt 1 500 x g Alternatively a 96 well PCR plate can be inserted into the Square well Block for elution 4 Centrifuge for 2 min at maximum speed gt 4 000 x g optimal 5 800 x g to collect the plasmid DNA Remove the Rack of Tube Strips containing eluted DNA and close them with Cap Strips for further storage MACHEREY NAGEL 04 2014 Rev 03 23 Plasmid DNA purification 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Cell pellet not properly resuspended Itis essential that the cell pellet is completely resuspended prior to lysis No cell clumps should be visible before addition of Lysis Buffer A2 If necessary increase number of mixing cycles or duration of shaking oo SDS in Buffer A2 precipitated eae SDS in Buffer A2 may precipitate upon storage If this happens li a white precipitate is visible at the bottom o
30. uffer A4 Concentrate 100 mL 200 mL 6 x 200 mL Elution Buffer AE 30 mL 125 mL 6 x 125 mL RNase A lyophilized 30 mg 60 mg 6 x 60 mg NucleoSpin Plasmid Binding 1 4 24 Plate white rings NucleoSpin Plasmid Filter Plate 1 4 24 viotel rings Culture Plate including Gas 1 4 24 permeable Foil Elution Plate including Self 1 4 24 adhering Foil MN Wash Plate 1 4 24 User manual 1 1 6 1 The kit for 24 x 96 preparations REF 740625 24 consists of 6 x REF 740625 4 2 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer AE 5 mM Tris HCl pH 8 5 4 MACHEREY NAGEL 04 2014 Rev 03 Plasmid DNA purification NucleoSpin 96 Plasmid Core Kit 4 x 96 preps 24 x 96 preps REF 740616 4 740616 24 Resuspension Buffer A1 150 mL 6x 150 mL Lysis Buffer A2 150 mL 6x 150 mL Neutralization Buffer A3 200 mL 6 x 200 mL Wash Buffer A4 Concentrate 2x 100 mL 12 x 100 mL Elution Buffer AE 125 mL 6 x 125 mL RNase A lyophilized 60 mg 6 x 60 mg NucleoSpin Plasmid Binding Plate 4 24 white rings NucleoSpin Plasmid Filter Plate 4 24 violet rings User manual 1 6 1 2 Reagents to be supplied by user 96 100 ethanol 1 The kit for 24 x 96 preparations REF 740616 24 consists of 6 x REF 740616 4 2 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer AE 5 mM Tris HCl pH 8 5 MACHEREY NAGEL 04 2014
31. x by inverting several times Incubate at room temperature 18 25 C for a maximum of 5 min with moderate shaking 300 rpm Note Do not vortex doing so will release contaminating chromosomal DNA from the cellular debris into the suspension Do not allow the lysis reaction to proceed for more than 5 minutes MACHEREY NAGEL 04 2014 Rev 03 19 NucleoSpin 96 Plasmid manual vacuum processing 4 Neutralize Add 350 uL Buffer A3 to the suspension For lysis in tubes close the culture tube and mix by inverting several times For lysis in plates either mix by pipetting up and down after addition of Buffer A3 or before loading to NucleoSpin Plasmid Filter Plate Optional Incubate on ice for 5 min for optimal formation of precipitate Prepare the NucleoVac 96 Vacuum Manifold Prepare manifold for filtration of crude lysates see page 18 Insert spacers labeled MTP Multi 96 Plate notched side up into the grooves located on the short sides of the manifold base Insert waste container into manifold base Place the NucleoSpin Plasmid Binding Plate white rings on top of the spacers Insert NucleoSpin Plasmid Filter Plate violet rings into the manifold lid and place the lid on the manifold base Close the manifold base with the manifold lid Close the vacuum manifold s valve 5 Transfer crude lysates onto the NucleoSpin Plasmid Filter Plate Transfer the crude lysates resulting from step 4 carefully and compl
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