Endotoxin-free plasmid DNA purification User manual
Contents
1. Endotoxin free plasmid DNA purification User manual NucleoBond 96 Xtra EF March 2014 Rev 04 MACHEREY NAGEL www mn net com SUN Endotoxin free plasmid DNA purification Table of contents 1 Components 1 1 Kit contents 1 2 Reagents consumables and equipment to be supplied by user 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 6 2 3 NucleoBond Xtra purification system 7 2 4 Endotoxins 9 2 4 1 Localization molecular structure and function of endotoxins 9 2 4 2 Quantification of endotoxins 9 2 4 3 Removal of endotoxins 9 2 5 Growth of bacterial cultures 10 2 5 1 Culture media and volume 10 2 5 2 Host strain and plasmid copy number 12 2 5 3 Chloramphenicol amplification of low copy plasmids 13 2 6 Lysate neutralization and LyseControl 13 3 Storage conditions and preparation of working solutions 14 4 Safety instructions 15 5 Protocols 17 5 1 NucleoBond 96 Xtra EF manual vacuum processing 17 5 2 NucleoBond 96 Xtra EF manual centrifuge processing 26 6 Appendix 28 6 1 Troubleshooting 28 6 2 Ordering information 31 6 3 Product use restriction warranty 32 MACHEREY NAGEL 03 2014 Rev 04 3 Endotoxin free plasmid DNA purification 1 Components 1 1 Kit contents NucleoBond 96 Xtra EF 1 x 96 preps 4 x 96 preps REF 740430 1 740430 4 Buffer RES EF 100 mL 2x 100 mL Buffer LYS EF 100 mL 2x 100 mL Buffer NEU EF 100 mL 2x 100 mL Buffer EQU EF2. contamination Insufficient washing If plasmid yield is low much unused binding capacity leads to tighter binding of RNA Double or triple washing steps Plasmid DNA does not perform well in downstream application Carry over of ethanol Silica membrane of the NucleoBond Finalizer Plate was not dry before elution or residual wash buffer droplets inside or outside the outlets Tap the NucleoBond Finalizer Plate onto a filter paper or soft tissue to soak up residual liquid Then dry NucleoBond Finalizer Plate under vacuum for at least 5 10 min EDTA in Elution Buffer TE EF EDTA may inhibit enzymatic reactions like DNA sequencing Use H O EF for elution DNA is irreversibly denatured Denatured plasmid DNA typically runs faster on agarose gels than supercoiled DNA Do not lyse the sample after addition of Buffer LYS EF for more than 5 min 30 MACHEREY NAGEL 03 2014 Rev 04 Endotoxin free plasmid DNA purification Problem Possible cause and suggestions Endotoxin level too high Too much cell mass was used Increase lysis buffer volume or reduce culture volume Inefficient endotoxin removal Make sure to wash two times with Buffer ENDO EF Contamination of DNA after purification Use only new pyrogen or endotoxin free consumables and plastics Endotoxins tend to stick to glass and are hard to remove If glass ware is used heat over night at 180 C to destroy endotoxins Autoclaving
3. 96 Xtra EF 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 32 MACHEREY NAGEL 03 2014 Rev 04 Endotoxin free plasmid DNA purification 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 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
4. Do not allow the lysis reaction to proceed for more than 5 minutes as prolonged exposure to alkaline conditions can irreversibly denature and degrade plasmid DNA and liberate contaminating chromosomal DNA 4 Neutralize Add 400 pL Buffer NEU EF to the suspension For lysis in tubes close the tubes and mix by inverting several times For lysis in plates mix by pipetting up and down very slowly and carefully after addition of Buffer NEU EF Mix the lysate until the blue color completely disappears to precipitate protein and chromosomal DNA and to completely renature plasmid DNA Optional Incubate on ice for 5 min for optimal formation of precipitate MACHEREY NAGEL 03 2014 Rev 04 21 NucleoBond 96 Xtra EF manual vacuum processing 5 Assemble vacuum manifold filtration set up Insert spacers labeled Square well Block notched side up into the grooves located on the short sides of the NucleoVac 96 Vacuum Manifold Place a new Square well Block inside the manifold Close the manifold with the manifold lid Place the NucleoBond Filter Plate on top of the manifold lid see page 21 6 Transfer crude lysates onto NucleoBond Filter Plate Mix the crude lysates from step 4 by pipetting the entire volume up and down once Transfer the lysates completely onto the NucleoBond Filter Plate 7 Clear crude lysate by vacuum filtration Apply vacuum of 0 4 to 0 6 bar 1 5 min If necessary press down th
5. devel oped by MACHEREY NAGEL for routine separation of different classes of nucleic acids such as oligonucleotides RNA and plasmids It consists of hydrophilic macroporous silica beads functionalized with MAE methyl amino ethanol The dense coating of this functional group provides a high positive charge density under acidic pH conditions that permits the negatively charged phosphate backbone of plasmid DNA to bind with high specificity Figure 1 anion exchanger CH group MAE Si gt spacer N N o o da KA i N ae oo y DNA backbone Figure 1 lonic interaction of the positively charged methyl hydroxyethyl amino MA group with the negative phosphate oxygen of the DNA backbone In contrast to the widely used DEAE diethylaminoethyl group the hydroxy group of MA can be involved in additional hydrogen bonding interactions with the DNA Due to a specialized manufacturing process which is strictly controlled and monitored the NucleoBond Xtra silica beads are uniform in diameter and contain particularly large pores These special properties allow optimized flow rates and sharp well defined elution profiles NucleoBond Xtra can separate distinct nucleic acid species from each other and from proteins carbohydrates and other unwanted cellular components over an exceptionally broad range of salt concentrations Figure 2 All contaminants from proteins to RNA and especially endotoxins are washed from th
6. manual vacuum processing 5 Protocols 5 1 NucleoBond 96 Xtra EF manual vacuum processing Before starting the preparation e Check that Buffer RES EF and 80 ethanol were prepared according to section 3 Protocol at a glance 1 Cultivate and harvest bacterial cells 1 5 5 mL cell culture 1 000 x g 10 min 2 Resuspend bacterial cells 400 pL RES EF Mix or shake 3 Lyse bacterial cells 400 uL LYS EF RT 2 5 min 4 Neutralize 400 pL NEU EF Mix or shake 5 Assemble vacuum manifold filtration set up see page 21 6 Transfer crude lysates onto NucleoBond Filter Plate light orange rings 7 Clear crude lysates by vacuum Apply vacuum 0 4 to 0 6 bar filtration 1 5 min 8 Assemble vacuum manifold Xtra purification set up see page 21 9 Equilibrate NucleoBond 900 pL EQU EF Xtra EF Plate Gravity flow 10 Load cleared lysates onto Gravity flow NucleoBond Xtra EF Plate Reduction of atmospheric pressure MACHEREY NAGEL 03 2014 Rev 04 17 NucleoBond 96 Xtra EF manual vacuum processing 11 Wash NucleoBond Xtra EF Plate 1 wash 900 pL ENDO EF Gravity flow EIS 900 pL ENDO EF Gravity flow EIZSJ 900 pL WASH EF Gravity flow 12 Assemble vacuum manifold Xtra elution set up see page 21 13 Elute DNA from NucleoBond Xtra EF 500 pL ELU EF Plate Gravity flow 14 Assemble vacuum manifold Finalizer purification set up see page 21 15
7. 100 mL 400 mL Buffer ENDO EF 200 mL 2 x 400 mL Buffer WASH EF 100 mL 500 mL Buffer ELU EF 60 mL 300 mL Buffer TE EF 125 mL 500 mL 80 EtOH Concentrate 50 mL 200 mL H 0 EF 30 mL 125 mL RNase A lyophilized 3 mg 2x6mg Culture Plate 1 4 with Gas permeable Foil Square well Block 2 8 Elution Plate U bottom with Self 1 4 adhering PE Foil for sealing NucleoBond Xtra EF Plate 1 4 NucleoBond Filter Plate 1 4 light orange rings NucleoBond Finalizer Plate 1 4 red rings MN Wash Plate 1 4 User manual 1 1 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 03 2014 Rev 04 Endotoxin free plasmid DNA purification 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol e isopropanol Consumables Disposable pipette tips Equipment Manual pipettors e NucleoVac 96 Vacuum Manifold 96 well plate or tube centrifuge for harvesting bacterial cells MACHEREY NAGEL 03 2014 Rev 04 5 Endotoxin free plasmid DNA purification 2 Product description 2 1 The basic principle The NucleoBond 96 Xtra EF procedure combines the most effective alkaline lysis with gravity flow anion exchange chromatography and fast vacuum filtration of isopropanol precipitated plasmid for extremely fast high throughput purification of endotoxin free plasmid DNA Bacteria are cultivated in the 96 well Culture Plate or glass tubes and harveste
8. L Bremer H Increased amplification of plasmids pBR322 and pBR327 by low concentrations of chloramphenicol DNA 5 539 544 1986 MACHEREY NAGEL 03 2014 Rev 04 13 Endotoxin free plasmid DNA purification 3 Storage conditions and preparation of working solutions Attention All kit components can be stored at room temperature 18 25 C and are stable for at least one year Storage of buffer LYS EF below 20 C may cause precipitation of SDS If salt precipitate is observed incubate buffer at 30 40 C for several minutes and mix well until all precipitate is redissolved completely Cool down to room temperature before use Before the first use of the NucleoBond 96 Xtra EF kit prepare the following Dissolve the lyophilized RNase A by adding 1 mL of Buffer RES EF 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 RES EF and shake well Note on the bottle the date of RNase A addition The final concentration of RNase A is 60 ug mL Store Buffer RES EF with RNase A at 4 C The solution will be stable at this temperature for at least 6 months Add indicated volume of 96 100 ethanol to the endotoxin free water in the bottles labeled 80 EtOH NucleoBond 96 Xtra EF 1 x 96 preps 4 x 96 preps REF 740430 1 740430 4 80 EtOH 50 mL 200 mL Concentrate Add 200 mL ethanol Add 800 mL ethanol to each bo
9. 3 2014 Rev 04 27 Endotoxin free plasmid DNA purification 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions No or low plasmid DNA yield Plasmid did not propagate Bacteria tend to lose their plasmids if not enough selective pressure by appropriate antibiotics is applied Use only fresh colonies and cultures for inoculation Always use enough fresh antibiotics in culture plates and media Incomplete cell lysis Cells are not completely resuspended Use only 1000 x g to pellet cells in order to obtain soft pellets Increase shaking shaking time or resuspend by pipetting up and down SDS precipitates in Lysis Buffer LYS EF when stored below 20 C Heat buffer to 30 40 C for several minutes until all precipitate is dissolved Cool down Buffer LYS EF to room temperature before use Too much cell mass was used for the given lysis buffer volumes Increase lysis buffer volumes or reduce culture volume for optimal lysis Resuspended cells are not mixed efficiently with lysis buffer Increase shaking during lysis incubation but do not vortex or pipette to avoid shearing of genomic DNA Alternatively seal the plate with self adhesive foil and invert the plate 5 6 times Be careful to avoid cross contamination when removing the foil Incomplete neutralization after cell lysis Increase shaking shaking time or mix by pipetting up and down very very gently Use wide bore pipette tips to avoid she
10. 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 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 a
11. N Wash Plate Waste Container Elution Plate U bottom Manifold base with spacers SQUARE WELL BLOCK inserted Manifold base with spacers SQUARE WELL BLOCK inserted Manifold base with spacers MTP MULTI 96 PLATE inserted 20 MACHEREY NAGEL 03 2014 Rev 04 NucleoBond 96 Xtra EF manual vacuum processing Detailed protocol Processing NucleoBond 96 Xtra EF under vacuum requires the NucleoVac 96 Vacuum Manifold Before starting the preparation Check that Buffer RES EF and 80 EtOH were prepared according to section 3 1 Cultivate and harvest bacterial cells Grow and harvest the bacterial cells as described in section 2 5 2 Resuspend bacterial cells Add 400 uL Buffer RES EF with RNase A to each sample Resuspend the cells completely by pipetting up and down or vortexing For an efficient lysis it is important that no clumps remain in suspension 3 Lyse bacterial cells Check Lysis Buffer LYS EF for precipitated SDS prior to use If a white precipitate is visible warm the buffer for several minutes at 30 40 C until precipitate is completely dissolved Cool buffer to room temperature 18 25 C before use Add 400 uL Buffer LYS EF to the suspension Incubate at room temperature for a maximum of 5 min with moderate shaking 300 rpm Warning Do not vortex as this will shear and release contaminating chromosomal DNA from cellular debris into the suspension
12. Precipitate plasmid DNA 350 uL isopropanol room temperature RT 5 min 16 Equilibrate NucleoBond Finalizer 1 mL TE EF Plate red rings Apply vacuum 0 2 to 0 4 bar 17 Load precipitated plasmid DNA onto Apply vacuum 0 2 to 0 4 bar NucleoBond Finalizer Plate 18 Wash NucleoBond Finalizer Plate 1 wash 1 mL 80 EtOH Apply vacuum 0 2 to 0 4 bar 2 wash 1 mL 80 EtOH Apply vacuum 0 2 to 0 4 bar 19 Assemble vacuum manifold Finalizer drying set up see page 21 Reduction of atmospheric pressure 18 MACHEREY NAGEL 03 2014 Rev 04 NucleoBond 96 Xtra EF manual vacuum processing 20 Dry NucleoBond Finalizer Plate Apply vacuum 0 4 to 0 6 bar 5 10 min 21 Assemble vacuum manifold Finalizer elution set up see page 21 22 Elute plasmid DNA from NucleoBond 100 200 pL TE EF or H O EF Finalizer Plate RT 1 3 min Apply vacuum max 0 4 bar 1 min Reduction of atmospheric pressure MACHEREY NAGEL 03 2014 Rev 04 19 NucleoBond 96 Xtra EF manual vacuum processing Setup of vacuum manifold Filtration setup NucleoBond Filter Plate light orange rings Square well Block Xtra Xtra purification elution setup setup NucleoBond Xtra EF Plate Waste Container Square well Block Finalizer Finalizer Finalizer purification drying elution setup setup setup NucleoBond Finalizer Plate red rings M
13. a thick cell wall Gram negative bacteria have a second membrane enclosing the inner membrane and only a thin cell wall The outer layer of this second membrane consists of amphiphilic lipopolysaccharides LPS also called endotoxins The structure of endotoxins can be divided into three domains 1 The hydrophobic Lipid A moiety is anchoring the LPS inside the membrane and confers the toxicity to endotoxins Its structure is highly conserved throughout all Gram negative bacteria 2 The hydrophilic inner core of the polysaccharide part of LPS the R antigen is a short sugar chain with a highly conserved sequence It is harboring a lot of negative charges and is thought to function as the main barrier against hydrophobic substances like antibiotics and detergents 3 The hydrophilic and extremely variable outer polysaccharide the O antigen is involved for example in cell adherence and interactions with the immune system of the host i e it is responsible for the immunological properties and virulence of the bacteria 2 4 2 Quantification of endotoxins Endotoxins can be measured in highly sensitive photometric tests Pyrochrome Associates of Cape Cod Inc and are expressed in endotoxin units EU For plasmid preparations the endotoxin level is given in EU per ug plasmid A concentration of 0 1 EU ug is usually considered endotoxin free 2 4 3 Removal of endotoxins Endotoxins are released from cells in small amounts during
14. aring of genomic DNA Alternatively seal the plate with self adhesive foil and invert the plate 10 15 times Be careful to avoid cross contamination when removing the foil Wrong elution buffers used Binding to the NucleoBond Xtra EF Plate is based on anion exchange chemistry Elution has to be done with the provided high salt ELU EF elution buffer TE EF or H O EF are for elution of the NucleoBond Finalizer Plate only 28 MACHEREY NAGEL 03 2014 Rev 04 Endotoxin free plasmid DNA purification Problem Possible cause and suggestions NucleoBond Xtra EF Plate was used under vacuum Incomplete binding and elution of DNA to anion exchange resin NucleoBond Xtra EF Plate must be run with gravity flow only No ethanol was added to 80 EtOH wash buffer DNA was eluted prematurely from the NucleoBond Finalizer Plate during the washing step Add appropriate amount of No or low 96 100 ethanol to wash buffer concentrate plasmid DNA yield Inefficient elution from NucleoBond Finalizer Plate continued When using other buffers for elution than the provided TE EF or H O EF make sure pH is higher than 7 The NucleoBond Finalizer Plate can only be used for plasmids ideally lt 15 kbp Larger constructs such as cosmids or BACs show much lower elution efficiency and tend to be damaged during the procedure Low copy plasmid was used Increase cell culture volume and lysis buffer volume See No or low plasmi
15. ccident 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 or strict liability arising in connection with MACHEREY NAGEL 03 2014 Rev 04 33 Endotoxin free plasmid DNA purification 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 conce
16. cell growth and in very large quantities upon cell death and lysis and thus also during plasmid purification Like intact cells the free LPS molecules induce inflammatory reactions of the mammalian immune system Therefore they have to be removed quantitatively from plasmid preparations to guarantee high transfection rates and high viability of transfected cells Due to their amphiphilic nature and their negative charge endotoxins behave like DNA and are co purified with most common plasmid purification systems Regular silica membrane kits with a purification procedure based on chaotropic salt lead to plasmid DNA with an endotoxin level of gt 1000 EU ug Anion exchange kits like NucleoBond Xtra reduce endotoxins to a level of lt 1 EU ug However since this may be still too high for successful transfection of very sensitive cells like primary or neuronal cells NucleoBond 96 Xtra EF was developed to reduce the endotoxin level to lt 0 05 EU ug plasmid DNA using a patented procedure MACHEREY NAGEL 03 2014 Rev 04 9 Endotoxin free plasmid DNA purification 2 5 Growth of bacterial cultures 2 5 1 Culture media and volume Yield and quality of plasmid DNA are highly dependent upon the type of culture media and antibiotics the bacterial host strain the plasmid type size and copy number and also on growth conditions For standard high copy plasmids LB Luria Bertani broth is recommended However due to the limited culture v
17. d DNA yield Incomplete neutralization after cell lysis NucleoBond Xtra EF or Cleared lysates were stored before loading onto the NucleoBond Filter Plate is Xtra EF Plate clogged More fine precipitate can form upon storage especially at lower temperatures Do not store cleared lysates but proceed immediately with the loading step Excessive mixing after addition of Buffer LYS EF or NEU EF Vortexing or pipetting can shear genomic DNA which is copurified with plasmid DNA Reduce number of mixing cycles reduce shaker speed and shaking time Use only wide Genomic DNA contamination bore pipette tips for pipetting gently up and down Genomic DNA was sheared during crude Iysate transfer to the NucleoBond Filter Plate Use wide bore pipette tips only Pipette very slowly MACHEREY NAGEL 03 2014 Rev 04 29 Endotoxin free plasmid DNA purification Problem Possible cause and suggestions Lysis time too long Genomic DNA and plasmid DNA start to degrade and will be rified Lysis time must not ex min Genomic DNA copurified Lysis time must not exceed 5 mi contamination i f continued Cell culture grown to late stationary or starvation phase Large amounts of dead cells are a source of genomic DNA fragments Reduce culture incubation time especially when using rich culture media No or low RNase A activity RNase A was not added to Buffer RES EF Dissolve RNase A RNA in Buffer RES EF and store buffer at 4 C
18. d by centrifugation The pelleted cells are resuspended and lysed according to a modified version of the Birnboim and Doly plasmid Mini prep protocol under alkaline conditions The resulting crude lysates are cleared under vacuum with a NucleoBond Filter Plate and loaded onto the NucleoBond Xtra EF Plate by gravity flow Extensive washing of the silica based anion exchange matrix removes RNA protein carbohydrates and endotoxins completely The DNA is eluted in a high salt buffer that requires a final clean up by isopropanol precipitation The precipitated plasmid DNA is loaded by vacuum filtration onto the NucleoBond Finalizer Plate washed and finally eluted in endotoxin free water or TE buffer 2 2 Kit specifications Kit specifications at a glance Parameter NucleoBond 96 Xtra EF Sample material 1 5 mL E coli culture Vector size lt 15 kbp lt 300 kbp without NucleoBond Finalizer Plate Typical yield 2 4 ug mL 1 5 mL LB TB in 96 well culture plates 10 50 g mL 5 mL LB TB in glass tubes A260 A280 1 80 1 95 Elution volume 100 200 uL Binding capacity 50 ug Endotoxin level lt 0 1 EU ug plasmid DNA Preparation time 120 min plate Birnboim H C amp Doly J 1979 Nucleic Acids Res 7 1513 1523 6 MACHEREY NAGEL 03 2014 Rev 04 Endotoxin free plasmid DNA purification 2 3 NucleoBond Xtra purification system NucleoBond Xtra Silica Resin is a patented silica based anion exchanger
19. does not inactivate endotoxins and is not recommended if the autoclave is also used for inactivation of bacterial waste Use only the endotoxin free buffers provided with the kit especially for preparation of 70 ethanol and TE EF or H2O EF for elution 6 2 Ordering information Product REF Pack of NucleoBond 96 Xtra EF 740430 1 1 x 96 preps 740430 4 4x 96 preps 740430 24 24 x 96 preps Buffer RES EF 740386 1000 1L without RNase A Buffer LYS EF 740387 1000 1L Buffer NEU EF 740388 1000 1L Buffer ENDO EF 740391 1000 1L Buffer WASH EF 740392 1000 1L Buffer ELU EF 740393 1000 1L RNase A lyophilized 740505 100 mg MACHEREY NAGEL 03 2014 Rev 04 31 Endotoxin free plasmid DNA purification Product REF Pack of 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 tubes each and 12 Cap Strips Cap Strips 740478 48 740478 24 288 MN Square well Block 740476 4 740476 24 24 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 NucleoBond
20. e NucleoBond Filter Plate slightly until the flow starts Adjust vacuum to establish a flow rate of 1 2 drops per second Release the vacuum when the crude lysate has passed the NucleoBond Filter Plate 8 Assemble vacuum manifold Xtra purification set up Discard the NucleoBond Filter Plate Take off the manifold lid Remove the Square well Block with cleared lysates Insert the waste container into the manifold base Close the manifold with the manifold lid Place the NucleoBond Xtra EF Plate on top of the manifold lid see page 21 9 Equilibrate NucleoBond Xtra EF Plate Attention step 9 13 are performed without applying any vacuum Load 900 uL Buffer EQU EF to each well and allow the buffer to pass the resin by gravity flow 10 Load cleared lysates onto NucleoBond Xtra EF Plate After Buffer EQU EF has completely run through load the cleared lysates from step 7 and allow the samples to pass the resin by gravity flow 11 Wash NucleoBond Xtra EF Plate After the cleared lysates have run through add 900 pL Buffer ENDO EF to each well Allow the buffer to pass the resin by gravity flow Reduction of atmospheric pressure 22 MACHEREY NAGEL 03 2014 Rev 04 NucleoBond 96 Xtra EF manual vacuum processing Repeat washing step with 900 uL Buffer ENDO EF Repeat washing step with 900 uL Buffer WASH EF 12 Assemble vacuum manifold Xtra elution set up Take off the manifold
21. e column The positive charge of the resin is neutralized by a pH shift to slightly alkaline conditions and pure plasmid DNA is eluted in a high salt elution buffer The purified nucleic acid products are suitable for use in the most demanding molecular biology applications including extremely sensitive transfections in vitro transcription automated or manual sequencing cloning hybridization and PCR MACHEREY NAGEL 03 2014 Rev 04 7 Endotoxin free plasmid DNA purification Plasmid DNA large constructs _ Single stranded DNA Double stranded DNA mRNA 168 235 rRNA gt 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 KCI Figure 2 Elution profile of NucleoBond Xtra Silica Resin at pH 7 0 The more interactions a nucleic acid can form between the phosphate backbone and the positively charged resin the later it is eluted with increasing salt concentration Large nucleic acids carry more charges than short ones and double stranded DNA more than single stranded RNA 8 MACHEREY NAGEL 03 2014 Rev 04 Endotoxin free plasmid DNA purification 2 4 Endotoxins 2 4 1 Localization molecular structure and function of endotoxins In contrast to Gram positive bacteria which have only one lipid bilayer membrane surrounded by
22. e bacteria grow much faster without the burden of a high copy plasmid they take over the culture rapidly and the plasmid yield goes down regardless of the cell mass Table 1 gives information on concentrations of commonly used antibiotics MACHEREY NAGEL 03 2014 Rev 04 11 Endotoxin free plasmid DNA purification Table 1 Information about antibiotics according to Maniatis Antibiotic Slock Solution Storage Working concentration concentration Ampicillin 50 mg mL in H O 20 C 20 50 ug mL Carbenicillin 50 mg mL in H O 20 C 20 60 ug mL Chloramphenicol 34 mg mL in EtOH 20 C 25 170 ug 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 ug mL Tetracycline 5 mg mL in EtOH 20 C 10 50 g mL 2 5 2 Host strain and plasmid copy number The quality of the plasmid DNA is mostly influenced by the E coli host strain used Whereas strains like DH5a or XL1 Blue usually produce high quality super coiled plasmid DNA other strains with high levels of endonuclease activity such as HB101 might yield lower quality plasmid giving poor results in downstream applications like enzymatic restriction or sequencing The type of plasmid especially its size and the origin of replication ori also has a crucial influence on DNA yield In general the larger the plasmid or the cloned insert the lower the expected DNA yield is due to a lower copy number Even a high copy co
23. fugation steps Required hardware and consumables A microtiter plate centrifuge which is able to accommodate the NucleoBond Filter Finalizer Plates stacked on a Square well Block or a Rack of Tube Strips bucket height 85 mm Rack of Tube Strips for elution see ordering information Additional MN Square well Block for collection of flow through see ordering information Before starting the preparation e Check that Buffer RES EF and 80 ethanol were prepared according to section 3 Cultivate harvest and lyse bacterial cells Follow steps 1 4 of the main protocol 5 1 for manual vacuum processing Clear crude lysate Place the NucleoBond Filter Plate on top of a MN Square well Block Mix the crude lysates by pipetting the entire volume up and down once Transfer the lysates completely onto the NucleoBond Filter Plate and centrifuge for 5 min at maximum speed Purify plasmid with NucleoBond Xtra EF Plate and precipitate DNA Follow steps 8 14 of the main protocol 5 1 for manual vacuum processing Equilibrate NucleoBond Finalizer Plate Place the NucleoBond Finalizer Plate on top of a MN Square well Block not included Add 1 mL Buffer TE EF to each well and centrifuge for 2 min at maximum speed Discard the flow through and place the NucleoBond Finalizer Plate back on top of the MN Square well Block 26 MACHEREY NAGEL 03 2014 Rev 04 NucleoBond Finalizer Plate manual cent
24. lid and the NucleoBond Xtra EF Plate Remove waste container and insert a new Square well Block Place the manifold lid with the inserted NucleoBond Xtra EF Plate back onto the manifold base see page 21 13 Elute DNA from NucleoBond Xtra EF Plate Load 0 5 mL Buffer ELU EF to each well and collect the eluted plasmid DNA by gravity flow 14 Assemble vacuum manifold Finalizer purification set up Insert spacers MTP Mult 96 Plate notched side up into the grooves located on the short sides of the manifold Insert waste container into the manifold base Insert the MN Wash Plate on the spacers inside the manifold base Close the manifold base with the manifold lid Place the NucleoBond Finalizer Plate on top of the manifold lid see page 21 15 Precipitate plasmid DNA Add 350 uL room temperature isopropanol to each well of the Square well Block with the eluted DNA to precipitate the plasmid Mix by repeated pipetting up and down Incubate for 5 min at room temperature 16 Equilibrate NucleoBond Finalizer Plate Attention Be sure to use the shortest vacuum time possible to avoid filtration of airborne bacteria onto the silica membrane Add 1 mL Buffer TE EF to each well and apply vacuum 0 2 to 0 4 bar until all liquid has passed the silica membrane Reduction of atmospheric pressure MACHEREY NAGEL 03 2014 Rev 04 23 NucleoBond 96 Xtra EF manual vacuum processing 17 Load precipitated plasmid DNA
25. lture plate In that case cultures are grown in up to 5 mL selective LB 16 24 h or rich medium 10 14 h at 37 C in an appropriate shaker 200 250 rpm 10 MACHEREY NAGEL 03 2014 Rev 04 Endotoxin free plasmid DNA purification 3 5 Cell growth OD 600 nm Plasmid yield ug 3 0 3 2 5 27 gt ke 2 0 E G S Q 1 5 E o 1 0 Q fo 0 5 0 0 T T T T T 1 0 5 10 15 20 25 30 Time h Figure 3 A 150 mL uniformly inoculated LB E coli DH5a pcDNA3 1 culture was split into a 96 well culture plate 1 5 mL well The cultures were shaken at 250 rpm and 37 C for up to 30 h Cultures were harvested in triplicate every hour and subjected to plasmid purification Cells can be harvested by centrifugation for 10 min at 1 000 x g In order to avoid cell pellets that are too tight to be easily dissolved higher g forces are not recommended For cultures grown in glass tubes cells can be harvested in the Culture Plate Therefore transfer only 1 5 mL of each culture to the Culture Plate centrifuge and discard the supernatant Repeat these steps to pellet the whole 5 mL culture Residual medium can be removed by tapping tubes or plate upside down on a clean paper sheet or soft tissue Bacterial cultures should be grown under antibiotic selection at all times to ensure plasmid propagation Without this selective pressure cells tend to lose a plasmid during cell division Sinc
26. n 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 verursachen MACHEREY NAGEL 03 2014 Rev 04 15 Endotoxin free plasmid DNA purification Precaution phrases P 210 P 233 P 234 P 261 P 280 P 302 352 P 304 341 P 305 351 338 P 332 313 P 333 313 P 337 313 P 342 311 P 363 P 390 P 403 235 P 406 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 Keep container tightly closed Beh lter dicht verschlossen halten Keep only in original container Nur im Originalbeh lter aufbewahren Avoid breathing dust Einatmen von Staub vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen 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 mi
27. nd Finalizer Plate back onto of the manifold lid See page 21 Reduction of atmospheric pressure 24 MACHEREY NAGEL 03 2014 Rev 04 NucleoBond 96 Xtra EF manual vacuum processing 22 Elute plasmid DNA from NucleoBond Finalizer Plate Add 100 200 uL Buffer TE EF or H O EF to each well The elution buffer should be dispensed carefully onto the center of the silica membrane Incubate the buffer on the membrane for 1 3 min at room temperature Apply vacuum of max 0 4 bar 1 min If necessary press down the NucleoBond Finalizer Plate slightly and collect the eluted DNA After the elution buffer has passed the wells release vacuum Note To increase final yield reapply the first eluate to the NucleoBond Finalizer Plate and elute a second time Alternatively use 100 200 uL of fresh Buffer TE EF H O EF for a second elution Furthermore heating elution buffer to 70 C can improve elution especially for DNA gt 5 10 kbp Remove the Elution Plate U bottom containing the eluted DNA and seal the plate with adhesive cover foil for further storage Reduction of atmospheric pressure MACHEREY NAGEL 03 2014 Rev 04 25 NucleoBond 96 Xtra EF manual centrifuge processing 5 2 NucleoBond 96 Xtra EF manual centrifuge processing To run the NucleoBond Filter Plate clearing the lysate and NucleoBond Finalizer Plate final clean up the vacuum procedure can be replaced by centri
28. nstruct based on a ColE1 ori can behave like a low copy vector if it contains a large or unfavorable insert In addition the ori itself influences the yield by a factor 10 100 For example plasmids based on pBR322 or pACYC cosmids or BACs are maintained at copy numbers less than 20 and can be as low as 1 copy per cell whereas vectors based on pUC pBluescript or pGEM can be present in several hundred copies per cell Therefore all the above mentioned factors should be taken into consideration if a particular DNA yield is required Culture volume and lysis procedures should be adjusted accordingly Maniatis T Fritsch EF Sambrook J Molecular cloning A laboratory manual Cold Spring Harbor Cold Spring New York 1982 12 MACHEREY NAGEL 03 2014 Rev 04 Endotoxin free plasmid DNA purification 2 5 3 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 OD o0 0 6 2 0 under selective conditions with an appropriate antibiotic Then add 170 g 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 bacterial culture
29. nutes Remove con tact 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 sp 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 rztlichen 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 Absorb spillage to prevent material damage Versch ttete Mengen aufnehmen um Materialsch den zu vermeiden Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort aufbewahren Store in a corrosive resistant container with a resistant inner liner In korrosionsbest ndigem Beh lter mit korrosionsbest ndiger AUskleidung aufbe wahren For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenblattern www mn net com 16 MACHEREY NAGEL 03 2014 Rev 04 NucleoBond 96 Xtra EF
30. o 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 34 MACHEREY NAGEL 03 2014 Rev 04
31. olume of 1 5 mL rich media such as 2x YT Yeast Tryptone TB Terrific Broth or CircleGrow can be used In such rich media bacteria grow faster reach the stationary phase much earlier and greater cell masses can be achieved compared to LB medium Cell cultures can either be grown directly in a Culture Plate which is provided with the kit or they can be cultivated in glass tubes The cell culture volume in the Culture Plate is limited to 1 2 1 5 mL of selective LB or rich medium in each well Exceeding the 1 5 mL limit can lead to cross contamination due to spillage during incubation A single bacterial colony should be seeded in each well and the Culture Plate covered with the Gas permeable Foil The cultures can then be grown in a suitable incubator at 37 C with vigorous shaking 200 400 rpm The Culture Plate can be fixed to the shaker with large size flask clamps for 2 L flasks or tape Cell growth is very slow under standard conditions due to bad oxygen supply and will take much longer to reach reasonable optical densities compared to oxygen saturated cultures Additionally even when the culture growth has slowed and seems to go stationary plasmid production is still in progress especially with high copy constructs Therefore prolonging incubation time from the typical 14 20 h to 30 35 h may achieve higher plasmid yields see Figure 3 To reach even higher yields well aerated glass tubes can be used instead of the 96 well cu
32. onto NucleoBond Finalizer Plate Load the mixture from step 15 to the NucleoBond Finalizer Plate and apply vacuum 0 2 to 0 4 bar until all liquid has passed the silica membrane 18 Wash NucleoBond Finalizer Plate Add 1 mL 80 EtOH to each well and apply vacuum 0 2 to 0 4 bar until all liquid has passed the silica membrane Repeat washing step with 1 mL 80 EtOH 19 Assemble vacuum manifold Finalizer drying step Take off the manifold lid and the NucleoBond Finalizer Plate Dry the outlets of the NucleoBond Finalizer Plate by placing it on a sheet or filter paper or soft tissue Remove the MN Wash Plate and the waste container Close the manifold with the manifold lid and place the NucleoBond Finalizer Plate back onto the manifold lid see page 21 20 Dry NucleoBond Finalizer Plate Apply vacuum 0 4 to 0 6 bar for 5 10 min to dry the membrane completely Run vacuum pump continuously Typically the adjusted vacuum is not reached at this step It is more important to have a continuous air flow to evaporate the ethanol Note The ethanol inhibits enzymatic reactions and has to be removed completely before eluting DNA Finally release the vacuum 21 Assemble vacuum manifold Finalizer elution set up Take off the manifold lid and the NucleoBond Finalizer Plate Insert the Elution Plate U bottom on the spacers inside the manifold base Close the manifold with the manifold lid Place the NucleoBo
33. rifuge processing Load precipitated plasmid DNA onto NucleoBond Finalizer Plate Load the mixture from step 3 to the NucleoBond Finalizer Plate and centrifuge for 2 min at maximum speed Discard the flow through and place the NucleoBond Finalizer Plate back on top of the MN Square well Block Wash NucleoBond Finalizer Plate Add 1 mL 80 EtOH to each well and centrifuge for 2 min at maximum speed Discard the flow through and place the NucleoBond Finalizer Plate back on top of the MN Square well Block Repeat washing step with 1 mL 80 EtOH Dry NucleoBond Finalizer Plate Centrifuge for 10 min at maximum speed Elute plasmid DNA from NucleoBond Finalizer Plate Place the NucleoBond Finalizer Plate on top of a Rack of Tube Strips not included Add 100 200 uL Buffer TE EF or H O EF to each well The elution buffer should be dispensed carefully onto the center of the silica membrane Incubate for 1 3 min at room temperature Centrifuge for 2 min at maximum speed To increase final yield reapply the first eluate to the NucleoBond Finalizer Plate and elute a second time Alternatively use 100 200 uL of fresh Buffer TE EF H O EF for a second elution Furthermore heating the elution buffer to 70 C can improve elution especially for DNA gt 5 10 kbp Note Do not use a microtiter plate as elution plate Microtiter plates may crack under centrifugation at gt 1 500 x g MACHEREY NAGEL 0
34. rning 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 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 0 24 21 969 270 e mail tech bio mn net com Trademarks Disclaimer DH5a is a registered trademark of Life Technologies Inc NucleoBond is a registered trademark of MACHEREY NAGEL GmbH amp Co KG pGEM is a registered trademark of Promega Pyrochrome is a registered trademark of Associates of Cape Cod Inc All used names and denotations can be brands trademarks or registered labels of their respective owner also if they are not special denotation T
35. s can be grown with only partial inhibition of protein synthesis under lower chloramphenicol concentrations 10 20 g mL resulting in a 5 to 10 fold greater yield of plasmid DNA Both methods show the positive 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 2 6 Lysate neutralization and LyseControl Proper mixing of the lysate with Neutralization Buffer NEU EF is of utmost importance for complete precipitation of SDS protein and genomic DNA Incomplete neutralization leads to reduced yields slower flow rates and potential clogging of the NucleoBond Filter Plate However released plasmid DNA is very vulnerable at this point and shaking too much or too strongly will damage the DNA Therefore do not vortex or shake but invert the mixture very gently until a fluffy off white precipitate has formed and the LyseControl has turned colorless throughout the lysate without any traces of blue color Maniatis T Fritsch EF Sambrook J Molecular cloning A laboratory manual Cold Spring Harbor Cold Spring New York 1982 Frenkel
36. ttle 14 MACHEREY NAGEL 03 2014 Rev 04 Endotoxin free plasmid DNA purification 4 Safety instructions The following components of the NucleoBond 96 Xtra EF kit contain hazardous con tents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features need not be 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 LYS EF Sodium hydroxide lt 2 Warning 290 315 234 280 Natriumhydroxid lt 2 gt Achtung 319 302 352 305 351 338 3324313 3374313 390 406 EQU EF Buffer salts ethanol Warning 226 210 233 WASH EF 5 20 403 235 Puffersalze Ethanol 5 20 Achtung ELU EF Buffer salts isopropanol Warning 226 319 210 233 280 10 15 305 351 338 Puffersalze Isopropanol Achtung 337 313 10 15 403 235 RNase A RNase A Iyophilized Danger 317 334 261 280 RNase A Iyophilisiert D 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 290 May be corrosive to metals Kann gegen ber Metallen korrosiv sein H 315 Causes skin irritation Verursacht Hautreizungen H 317 May cause an allergic ski
Download Pdf Manuals
Related Search