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Plasmid DNA purification - Lab Supplies Scientific

1. NucleoBond Pellet Rec OD o9 OD OD OD OD Xtra wet ODV weight 2 4 6 8 10 Midi 1 59 800 400 mL 200 mL 133 mL 100 mL 80 mL 1200 Maxi 4 5 g 2400 i 600 mL 400 mL 300 mL 240 mL For higher yields it is advantageous to increase the cell culture and lysis buffer volumes even more e g by factor 3 5 In this case additional lysis buffer can be ordered separately see ordering information Furthermore a centrifuge should be used for lysate clarification instead of the provided NucleoBond Xtra Column Filters since their capacity for precipitate is limited Alternatively chloramphenicol amplification can be considered to increase the plasmid copy number see section 4 4 16 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification 4 7 Cell lysis The bacterial cell pellet is resuspended in Buffer RES and lysed by a sodium hydroxide SDS treatment with Buffer LYS Proteins as well as chromosomal and plasmid DNA are denatured under these conditions RNA is degraded by DNase free RNase A Neutralization Buffer NEU containing potassium acetate is then added to the lysate causing SDS to precipitate as KDS potassium dodecyl sulfate and pulling down proteins chromosomal DNA and other cellular debris The potassium acetate buffer also neutralizes the lysate Plasmid DNA can revert to its native super coiled structure and remains in solution The NucleoBond X
2. Double stranded DNA le mRNA 16S 23S rRNA 5S rRNA Compound class ee tRNA p Proteins dyes polysaccharides metabolites trinucleotides rRNA large constructs Plasmid DNA Absorbance at 260 nm 0 0 5 1 1 5 2 Salt concentration for elution M KCI Figure 2 Elution profile of NucleoBond Xtra Silica Resin at pH 7 0 The more interactions a nucleic acid can form between 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 12 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification 4 3 Growth of bacterial cultures Yield and quality of plasmid DNA highly depend on the type of culture media and antibiotics the bacterial host strain the plasmid type size and copy number but also on the growth conditions For standard high copy plasmids LB Luria Bertani medium is recommended The cell culture should be incubated at 37 C with constant shaking 200 250 rpm preferably 12 16 h over night Use flasks of at least three or four times the volume of the culture volume to provide a growth medium saturated with oxygen Alternatively rich media like 2xYT Yeast Tryptone TB Terrific Broth or CircleGrow can be used In this case bacteria grow faster reach the stationary phase much earlier than in LB medium lt 2h an
3. 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 doctor phy sician Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM oder 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 Sicherheitsdatenbl ttern www mn net com MACHEREY NAGEL 03 2012 Rev 10 27 NucleoBond Xtra Midi Maxi 7 NucleoBond Xtra plasmid purification The following section includes the protocols for high copy and low copy plasmid purification as well as for concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers 7 1 High copy plasmid purification Midi Maxi Prepare a starter culture Inoculate a 3 5 mL starter culture of LB medium with a single colony picked from a freshly streaked agar plate Ma
4. Pellet the cells by centrifugation at 4 500 6 000 x gfor 210 min at 4 C and dis card the supernatant completely Note It is of course possible to use larger culture volumes for example if a large amount of low copy plasmid is needed see section 4 6 for more information In this case increase RES LYS and NEU buffer volumes proportionally in steps 4 5 and 7 Additional Iysis buffer volumes might have to be ordered separately see ordering information for NucleoBond Xtra Buffer Set I section 8 2 Use a centrifuge for the lysate clarification rather than the NucleoBond Xtra Column Filters 34 MACHEREY NAGEL 03 2012 Rev 10 NucleoBond Xtra Midi Maxi 2 4 Resuspension Buffer RES Resuspend the cell pellet completely in Resuspension Buffer RES RNase A by pipetting the cells up and down or vortexing the cells For an efficient cell lysis it is important that no clumps remain in the suspension Note Increase RES buffer volume proportionally if more than the recommended cell mass is used see section 4 7 for information on optimal cell lysis and section 4 8 regarding difficult to lyse strains 5 Cell lysis Buffer LYS Check Lysis Buffer LYS for precipitated SDS prior to use If a white precipitate is visible warm the buffer for several minutes at 30 40 C until precipitate is dissolved completely Cool buffer down to room temperature 18 25 C Add Lysis Buffer LYS
5. Plasmid DNA purification User manual NucleoBond Xtra Midi NucleoBond Xtra Maxi NucleoBond Xtra Midi Plus NucleoBond Xtra Maxi Plus March 2012 Rev 10 MACHEREY NAGEL MN Plasmid DNA purification NucleoBond Xtra Midi Maxi Protocol at a glance Rev 10 1 3 Cultivate and harvest bacterial cells 4 500 6 000 x g 4 C 15 min 7 Neutralization 8 Clarification and loading of the lysate 4 5 Cell lysis High copy low copy High copy low copy Important 8mL 16mL Buffer RES 12 mL 24 mL Buffer RES Check Buffer LYS for 8mL 16 mL Buffer LYS 12 mL 24 mL Buffer LYS precipitated SDS RT 5 min RT 5 min 6 Equilibration of the column and filter 12 mL 25 mL Buffer EQU Buffer EQU 8 mL 16 mL Buffer NEU Invert the tube 3 times Load lysate on NucleoBond Xtra Column Filter 12 mL 24 mL Buffer NEU 9 1 Washing 10 Discard NucleoBond Xtra Column Filter 5 mL 1 Buffer EQU Discard NucleoBond Xtra Column Filter u 15 mL Buffer EQU A Discard NucleoBond Xtra Column Filter 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 11 2 Washing gmk 25 mL l Buffer WASH Buffer WASH 12 Elution 5mL 15 mL Buffer ELU Buffer ELU 13 Precipitation NucleoBond NucleoBond eoBond eoBond Xtra Midi Xtra Midi Plus P 3 5 mL 3 5 mL 10
6. Plasmid DNA purification 8 Appendix 8 1 Troubleshooting If you experience problems with reduced yield or purity it is recommended to check which purification step of the procedure is causing the problem First the bacterial culture has to be checked for sufficient growth OD in the presence of an appropriate selective antibiotic Table 1 section 4 3 Second aliquots of the cleared lysate the flow through the combined washing steps Buffer EQU and Buffer WASH and the eluate should be kept for further analysis by agarose gel electrophoresis Refer to Table 6 to choose a fraction volume yielding approximately 5 ug of plasmid DNA assuming 250 ug and 1000 ug were loaded onto the NucleoBond Xtra Midi and Maxi Column respectively Precipitate the nucleic acids by adding 0 7 volumes of isopropanol centrifuge the sample wash the pellet using 70 ethanol centrifuge again remove supernatant air dry for 10 minutes dissolve the DNA in 100 uL TE buffer pH 8 0 and run 20 uL on a 1 agarose gel Table 6 NucleoBond Xtra eluate volumes required for an analytical check Volume required pL Sample Purification step Midi Maxi Cleared lysate of protocol step 8 a an m Column flow through 500 200 after protocol step 8 Wash flow through u after protocol step 9 and 11 250 200 Eluate x after protocol step 12 U 100 The exemplary gel picture Figure 6 will help you to address the specific q
7. inoculating loop culture tubes and flasks 37 C shaking incubator and centrifuge with rotor and tubes or bottles for harvesting cells Refrigerated centrifuge capable of reaching 5 000 x g with rotor for the appropriate centrifuge tubes or bottles not necessary for NucleoBond Xtra Midi Maxi Plus kits Centrifugation tubes or vessels with suitable capacity for the volumes specified in the respective protocol NucleoBond Xtra Combi Rack see ordering information or equivalent holder MACHEREY NAGEL 03 2012 Rev 10 T Plasmid DNA purification 2 Kit specifications NucleoBond Xtra kits are suitable for ultra fast purification of plasmids cosmids and very large constructs P1 constructs BACs PACs ranging from 3 kbp up to 300 kbp For preparation of working solutions and storage conditions see section 5 NucleoBond Xtra Columns are polypropylene columns containing Nucleo Bond Xtra Silica Resin packed between two inert filter elements The columns are available in Midi and Maxi sizes with typical DNA yields of 250 ug and 1000 ug respectively All NucleoBond Xtra Columns are resistant to organic solvents such as alcohol chloroform and phenol and are also suitable for buffers containing denaturing agents like formamide urea or common detergents like Triton X 100 or NP 40 NucleoBond Xtra Silica Resin can be used over a wide pH range pH 2 5 8 5 and can remain in contact with buffers for several
8. additional buffer has to be ordered separately for routine purification of low copy plasmids see ordering information Prepare a starter culture Inoculate a 3 5 mL starter culture of LB medium with a single colony picked from a freshly streaked agar plate Make sure that plate and liquid culture contain the appropriate selective antibiotic to guarantee plasmid propagation see section 4 3 for more information Shake at 37 C and 300 rpm for 8 h oa Prepare a large overnight culture Note To utilize the entire large binding capacity of the NucleoBond Xtra Columns it is important to provide enough plasmid DNA For the standard low copy procedure the culture volumes were doubled compared to the high copy vector protocol However due to a plasmid content that is 10 100 times lower this might be insufficient If you need large amounts of low copy plasmids further increase the culture volume by factor 3 5 The recommended culture volumes below are calculated for a final OD o of around 4 see section 4 6 for more information Inoculate an overnight culture by diluting the starter culture 1 1000 into the given volumes of LB medium also containing the appropriate selective antibiotic Grow the culture overnight at 37 C and 300 rpm for 12 16 h 200 mL 600 mL Harvest bacterial cells Measure the cell culture OD and determine the recommended culture volume V mL 800 OD V mL 2400 OD
9. 1 ug uLl 1 6 ug uL 1 2 ug uL 1 0 ug uL 3 10 40 70 85 90 90 500 ug 1 3 ug uL 1 4 ug uL 1 0 ug uL 0 8 ug uL 0 6 ug uL 0 5 ug uL 15 45 70 80 85 90 0 4 ug uL 0 3 ug uL 0 2 ug L 0 1 ug uL 0 1 ug L 0 1 ug L 100 pg DNA recovery DNA concentration 22 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification 4 13 Determination of DNA yield and quality The yield of a plasmid preparation should be estimated prior to and after the isopropanol precipitation in order to calculate the recovery after precipitation and to find the best volume to dissolve the pellet in Simply use either NucleoBond Xtra Elution Buffer ELU or the respective low salt buffer as a blank in your photometric measurement The nucleic acid concentration of the sample can be calculated from its UV absorbance at 260 nm where an absorbance of 1 1 cm path length is equivalent to 50 ug DNA mL Note that the absolute measured absorbance should lie between 0 1 and 0 7 in order to be in the linear part of Lambert Beer s law Dilute your sample in the respective buffer if necessary The plasmid purity can be checked by UV spectroscopy as well A ratio of A A between 1 80 1 90 and A A around 2 0 indicates pure plasmid DNA An A A ratio above 2 0 is a sign for too much RNA in your preparation an A A ratio below 1 8 indicates protein contamination Plasmid quali
10. 2012 Rev 10 Plasmid DNA purification 4 NucleoBond Xtra plasmid purification system 4 1 Basic principle The bacterial cells are lysed by an optimized set of newly formulated buffers based on the NaOH SDS lysis method of Birnboim and Doly After equilibration of the NucleoBond Xtra Column together with the corresponding NucleoBond Xtra Column Filter the entire lysate is loaded by gravity flow and simultaneously cleared by the specially designed column filter Plasmid DNA is bound to the NucleoBond Xtra Silica Resin After an efficient washing step the plasmid DNA is eluted precipitated and easily dissolved in any suitable buffer e g low salt buffer or water for further use 4 2 NucleoBond Xtra anion exchange columns NucleoBond Xtra is a patented silica based anion exchange resin developed by MACHEREY NAGEL It is developed for routine separation of different classes of nucleic acids like oligonucleotides RNA and plasmids NucleoBond Xtra Silica Resin consists of hydrophilic macroporous silica beads functionalized with MAE methyl amino ethanol The dense coating of this functional group provides a high overall positive charge density under acidic pH conditions that permits the negatively charged phosphate backbone of plasmid DNA to bind with high specificity Figure 1 anion exchanger CH group MAE si gt spacer NS o OH CH a O LN p e DNA backbone
11. 5 mL 10 5 mL Isopropanol Isopropanol Isopropanol Isopropanol RT 2 min RT 2 min 5 15 000 x g Load 15 000 x g Load NucleoBond 4 C 30 min NucleoBond 4 C 30 min Finalizer Large Finalizer 14 Wash and dry DNA 2 mL 2 mL 5 mL 5 mL pellet 70 ethanol 70 ethanol 70 ethanol 70 ethanol 5 15 000 x g 5 15 000 x g RT 5 min RT 5 min 5 10 min 23 x air until dry 10 15 min gt 6 x air until dry 15 Reconstitute DNA Appropriate 200 800 uL Appropriate 400 1000 uL volume of Buffer TRIS volume of Buffer TRIS TE buffer TE buffer Plasmid DNA purification Table of contents 1 Components 1 1 Kit contents 1 2 Reagents and equipment to be supplied by user Kit specifications About this user manual NucleoBond Xtra plasmid purification system 4 1 Basic principle 4 2 NucleoBond Xtra anion exchange columns 4 3 Growth of bacterial cultures 4 4 Chloramphenicol amplification of low copy plasmids 4 5 Culture volume for high copy plasmids 4 6 Culture volume for low copy plasmids 4 7 Cell lysis 4 8 Difficult to Iyse strains 4 9 Setup of NucleoBond Xtra Columns 4 10 Filtration and loading of the lysate 4 11 Washing of the column 4 12 Elution and concentration of plasmid DNA 4 13 Determination of DNA yield and quality 4 14 Convenient stopping points Storage conditions and preparation of working solutions Safety instructions risk and safety phrases 6 1 Risk and safety phrases 6 2 GHS classification Nucle
12. DNA 8 kbp was loaded onto a NucleoBond Finalizer and eluted two fold with increasing volumes of TE buffer The NucleoBond Finalizer is designed to hold a maximum of 500 ug DNA and is therefore ideally suited to be used in combination with NucleoBond Xtra Midi Maximum DNA recovery can be achieved by using gt 600 uL of elution buffer For a higher concentration experienced users can lower the elution buffer volume to 400 200 uL Table 4 gives an overview about recovery and concentration of different amounts of plasmid DNA loaded onto a NucleoBond Finalizer DNA was eluted two fold with increasing volumes of TE Please refer to this tables to select an elution buffer volume that meets your needs best Table 4 DNA recovery and concentration for the NucleoBond Finalizer Elution volume 100 pL 200 uL 400 uL 600 uL 800 pL 1000 pL 35 60 75 75 75 2 5 yg uL 2 3 ug L 0 8 ug uL 0 6 pg uL 0 5 ug L 40 65 75 80 80 80 1 9 ug uL 1 1 ug uL 0 6 ug uL O Aug ulL 0 3 ug uL 0 2 ug uL 45 70 80 85 85 85 0 7 ug uL O4yg UL 0 2 ug L 0 1 ug uL 0 1 ug L 0 1 ug L 30 75 85 90 90 90 0 3 ug uL O 2ug uL O 1 ug L 0 1 ug uL 0 1 ug uL lt 0 1 pg L 70 1 2 ug uL 500 ug 250 ug Loaded DNA 100 pg 50 ug DNA recovery DNA concentration MACHEREY NAGEL 03 2012 Rev 10
13. Figure 1 lonic interaction of the positively charged methyl hydroxyethyl amino group with the negative phosphate oxygen of the DNA backbone In contrast to the widely used DEAE diethylaminoethyl group the hydroxy group of methyl hydroxyethyl amin can be involved in additional hydrogen bonding interactions with the DNA Birnboim H C and Doly J 1979 Nucl Acids Res 7 1513 1523 MACHEREY NAGEL 03 2012 Rev 10 11 Plasmid DNA purification Due to a specialized manufacturing process that is strictly controlled and monitored the NucleoBond Xtra silica beads are uniform in diameter and contain particularly large pores These special properties allow optimized flow rates and sharp well defined elution profiles NucleoBond Xtra can separate distinct nucleic acid species from each other and from proteins carbohydrates and other unwanted cellular components over an exceptionally broad range of salt concentrations Figure 2 All contaminants from proteins to RNA are washed from the column the positive charge of the resin is neutralized by a pH shift to slightly alkaline conditions and pure plasmid DNA is eluted in a high salt elution buffer The purified nucleic acid products are suitable for use in the most demanding molecular biology applications including transfection in vitro transcription automated or manual sequencing cloning hybridization and PCR Plasmid DNA large constructs Single stranded DNA
14. Setup for elution 18 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification 4 10 Filtration and loading of the Iysate After the alkaline lysis the sample has to be cleared from cell debris and precipitate to ensure high plasmid purity and a fast column flow rate This is achieved by passing the solution through a NucleoBond Xtra Column Filter which is provided already inserted into the NucleoBond Xtra Column NucleoBond Xtra NucleoBond Xtra Midi Maxi NucleoBond Xtra Column Filter NucleoBond Xtra Column The NucleoBond Xtra Column Filters are designed to eliminate the centrifugation step after alkaline Iysis They are pre wet during column equilibration and allow a time saving simultaneous clearing of bacterial lysate and loading of the NucleoBond Xtra Column Compared to lysate clearing by centrifugation or syringe filters the NucleoBond Xtra Column Filter furthermore avoids shearing of large DNA constructs such as PACs or BACs by the gentle depth filter effect filtration occurs on the surface of the filter as well as inside the filter matrix Its special material and design lead to very rapid passage of the lysate through the filter and even very large lysate volumes can be applied without the risk of clogging This is especially important for low copy plasmid purification for example However if more than the recommended cell mass see section 4 5 Table 2 section 4 6 Table 3 was lysed it might
15. be advantageous to use a centrifuge for lysate clarification rather than the provided column filters due to their limited precipitate capacity 4 11 Washing of the column The high salt concentration of the lysate prevents proteins and RNA from binding to the NucleoBond Xtra Column see section 4 2 Figure 2 However to remove all traces of contaminants and to purge the dead volume of the NucleoBond Xtra Column Filters it is important to wash the column and the filter in two subsequent washing steps First apply Equilibration Buffer EQU to the funnel rim of the filter to wash all residual lysate out of the filter onto the column Do not just pour the buffer inside the filter Then pull out and discard the column filter or remove the filter by turning the column upside down It is essential to wash the NucleoBond Xtra Column without filter for a second time with Wash Buffer WASH This ensures highest yields with best achievable purity MACHEREY NAGEL 03 2012 Rev 10 19 Plasmid DNA purification 4 12 Elution and concentration of plasmid DNA Elution is carried out under high salt conditions and by a shift of pH from 7 0 to 9 0 Under these alkaline conditions the positive charge of the anion exchange resin is neutralized and plasmid DNA is released For any downstream application it is necessary to precipitate the DNA and to remove salt and all traces of alcohol since they disturb or inhibit enzymatic activity needed for restric
16. hours without any change in its chromatographic properties The NucleoBond Xtra Column Filters are specially designed depth filters that fit into the NucleoBond Xtra Columns The filters are inserted ready to use in the NucleoBond Xtra Columns and allow a time saving simultaneous clearing of bacterial lysate and loading of cleared lysate onto the NucleoBond Xtra Column Furthermore the use of the column filters avoids the time consuming centrifugation step for lysate clearing The NucleoBond Xtra Column Filters allow complete removal of precipitate even with large lysate volumes without clogging and avoid shearing of large DNA constructs such as PACs or BACs by the gentle depth filter effect The NucleoBond Xtra Midi Plus and NucleoBond Xtra Maxi Plus kits additionally contain the NucleoBond Finalizers and NucleoBond Finalizers Large respectively These tools for a fast concentration and desalination of eluates are suitable for most plasmids and cosmids ranging from 2 50 kbp with recovery efficiencies from 40 90 depending on elution volume NucleoBond Finalizer is a polypropylene syringe filter containing a special silica membrane The NucleoBond Finalizer provides a binding capacity of 500 ug whereas the NucleoBond Finalizer Large can hold up to 2000 ug plasmid DNA Due to the small dead volumes of the NucleoBond Finalizers the plasmid DNA can be eluted with a concentration up to 3 ug uL see section 4 12 T
17. 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 MACHEREY NAGEL 03 2012 Rev 10 49 Plasmid DNA purification 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 ma
18. recovery after precipitation 13 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Vortex thoroughly Centrifuge at 5 000 x g for 15 min at sroom temperature preferably at 15 000 x g for 30 min at 4 C Carefully discard the supernatant 3 5 mL 10 5 mL 32 MACHEREY NAGEL 03 2012 Rev 10 NucleoBond Xtra Midi Maxi 14 Wash and dry DNA pellet Add room temperature 70 ethanol to the pellet 2mL 5 mL Centrifuge at gt 5 000 x g preferably 15 000 x gfor 5 min at room temperature 18 25 C Carefully remove ethanol completely from the tube with a pipette tip Allow the pellet to dry at room temperature 18 25 C Note Plasmid DNA might be harder to dissolve when over dried 15 Reconstitute DNA Dissolve the DNA pellet in an appropriate volume of buffer TE or sterile H O Depending on the type of centrifugation tube dissolve under gentle pipetting up and down or constant spinning in a sufficient amount of buffer for 10 60 min 3D shaker Determine plasmid yield by UV spectrophotometry Confirm plasmid integrity by agarose gel electrophoresis see section 4 13 MACHEREY NAGEL 03 2012 Rev 10 33 NucleoBond Xtra Midi Maxi 7 2 Low copy plasmid purification Midi Maxi The lysis buffer volumes provided in the kit are adjusted for high copy plasmid purification Therefore
19. the NucleoBond Xtra Column Filter and Nu cleoBond Xtra Column with Equilibration Buffer EQU Apply the buffer to the funnel shaped rim of the filter and make sure it is washing out the lysate which is remaining in the filter Omitting this step or just pouring the buffer directly inside the funnel may reduce plasmid yield 10 Discard column filter Either pull out the NucleoBond Xtra Column Filter or discard it by turning the column upside down MACHEREY NAGEL 03 2012 Rev 10 31 NucleoBond Xtra Midi Maxi 11 p R Wash column Buffer WASH Wash the NucleoBond Xtra Column with Wash Buffer WASH It is important to remove the column filter before applying the washing buffer to avoid low purity 12 Elution Buffer ELU Elute the plasmid DNA with Elution Buffer ELU Collect the eluate in a 15 mL or 50 mL centrifuge tube not provided Note Preheating Buffer ELU to 50 C prior to elution may improve yields for large constructs such as BACs Proceed with step 13 for the centrifugation protocol after isopropanol precipitation or continue with section 7 3 for plasmid concentration and desalination by using the NucleoBond Finalizer NucleoBond Xtra Midi Plus or NucleoBond Finalizer Large NucleoBond Xtra Maxi Plus Optional Determine plasmid yield by UV spectrophotometry in order to adjust desired concentration of DNA in step 15 and calculate the
20. 21 Plasmid DNA purification 100 3 0 90 80 a ir F2 5 70 20 60 50 F1 5 40 30 20 Recovery Concentration Recovery Concentration ug uL F0 5 T T T T T 0 0 0 200 400 600 800 1000 Elution volume uL Figure5 Final DNA recovery and concentration after NucleoBond Finalizer Large application A NucleoBond Xtra Maxi eluate containing 1000 ug plasmid DNA 8 kbp was loaded onto a NucleoBond Finalizer Large and eluted two fold with increasing volumes of TE buffer NucleoBond Xtra Maxi eluates are easily concentrated with a NucleoBond Finalizer Large which is able to bind up to 2000 ug plasmid DNA Maximum DNA recovery can be achieved by using gt 800 uL of elution buffer For a higher concentration experienced users can lower the elution buffer volume to 600 400 jL Table 5 gives an overview about recovery and concentration of different amounts of plasmid DNA loaded onto a NucleoBond Finalizer Large DNA was eluted two fold with increasing volumes of TE Please refer to this tables to select an elution buffer volume that meets your needs best Table 5 DNA recovery and concentration for the NucleoBond Finalizer Large Elution volume 100 pL 200 uL 400 uL 600 uL 800 pL 1000 pL 1500 ug 1 9 ug uL 3 2 ug uL 2 9 ug uL 2 2 ug uL 1 7 ug uL 1 4 ug uL s 5 35 70 85 90 90 a 1000 ug 5 ESE 2 5 ug uL 2
21. 25 C 6 7 Add Lysis Buffer LYS to the suspension Mix gently by inverting the tube 5 times Do not vortex as this will shear and release contaminating chromosomal DNA from cellular debris into the suspension Incubate the mixture at room temperature 18 25 C for 5 min Warning Prolonged exposure to alkaline conditions can irreversibly denature and degrade plasmid DNA and liberate contaminating chromosomal DNA into the lysate Note Increase LYS buffer volume proportionally if more than the recommended cell mass is used see section 4 7 for information on optimal cell lysis If you are performing a Midi prep to purify plasmid DNA you will find volumes or incubation times in the white boxes For Maxi preps please refer to the black boxes The name of the buffer incubation times repeats or important handling steps are emphasized in bold type within the instruction Additional notes or optional steps are MACHEREY NAGEL 03 2012 Rev 10 9 Plasmid DNA purification printed in italic The exclamation point marks information and hints that are essential for a successful preparation In the example shown above you are asked to check the Lysis Buffer LYS prior to use and then to lyse the resuspended cell pellet in 8 mL of Buffer LYS when performing a Midi prep and in 12 mL for a Maxi prep Follow the handling instructions exactly and note the given hints for protocol alterations 10 MACHEREY NAGEL 03
22. 7 for larger culture volumes and adjusted lysis buffer volumes Xtra Column is blocked or Make sure to mix well after neutralization to completely very slow precipitate SDS and chromosomal DNA Lysate was not cleared completely Use NucleoBond Xtra Column Filter or centrifuge at higher speed or for a longer period of time Precipitates occur during storage Clear lysate again before loading the column Lysis treatment was too harsh Make sure not to lyse in Buffer LYS for more than 5 min Genomic can n ew n Lysate was mixed too vigorously or vortexed after lysis plasmid DNA Invert tube for only 5 times Do not vortex after addition of Buffer LYS Use larger tubes or reduce culture volumes for easier mixing MACHEREY NAGEL 03 2012 Rev 10 43 Plasmid DNA purification Problem RNA conta mination of plasmid DNA Possible cause and suggestions RNase digestion was inefficient RNase was not added to Buffer RES or stored improperly Add new RNase to Buffer RES See section 8 2 for ordering information pH or salt concentration of wash buffer is too low Check RNA content in the wash fractions see Figure 6 Keep all buffers tightly closed Check pH of Buffer EQU pH 6 5 and WASH pH 7 0 and adjust with HCI or NaOH if necessary Wash step with Buffer WASH was not sufficient Double or triple washing step with Buffer WASH Additional Buffer WASH can be ordered separately see ordering information Low p
23. able 4 and 5 for dependence of concentration on elution volume All NucleoBond Finalizers are resistant to organic solvents such as alcohol chloroform and phenol and are free of endotoxins MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification 3 About this user manual The following section 4 provides you with a detailed description of the NucleoBond Xtra purification system and important information about cell growth cell lysis and the subsequent purification steps Sections 5 and 6 inform you about storage buffer preparation and safety instructions First time users are strongly advised to read these chapters thoroughly before using this kit Experienced users can directly proceed with the purification protocols section 7 or just use the Protocol at a glance for a quick reference Section 7 includes the protocols for high copy and low copy plasmid purification as well as for the concentration of NucleoBond Xtra eluates with the NucleoBond Finalizer This part of the protocol is also available at www mn net com in French and German Each procedural step in the purification protocol is arranged like the following example taken from section 7 1 5 Cell lysis Buffer LYS Check Lysis Buffer LYS for precipitated SDS prior to use If a white precipitate is visible warm the buffer for several minutes at 30 40 C until precipitate is dissolved completely Cool buffer down to room temperature 18
24. ase OD 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 the cell culture can be grown with only partial inhibition of protein synthesis under low chloramphenicol concentrations 10 20 g mL resulting in a 5 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 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 14 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA
25. ct 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 N 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 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 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
26. d higher cell masses can be reached However this does not necessarily yield more plasmid DNA Overgrowing a culture might lead to a higher percentage of dead or starving cells and the resulting plasmid DNA might be partially degraded or contaminated with chromosomal DNA To find the optimal culture conditions the culture medium and incubation times have to be optimized for each host strain plasmid construct combination individually Cell cultures should be grown under antibiotic selection at all times to ensure plasmid propagation Without this selective pressure cells tend to lose a plasmid during cell division Since bacteria grow much faster without the burden of a high copy plasmid they take over the culture rapidly and the plasmid yield goes down regardless of the cell mass Table 1 gives information on concentrations of commonly used antibiotics Table 1 Information about antibiotics according to Maniatis Antibiotic Stock solution Storage Working concentration concentration Ampicillin 50 mg mL in H O 20 C 20 60 pg 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 g mL Tetracycline 5 mg mL in EtOH 20 C 10 50 ug mL Carbenicillin 50 mg mL in H O 20 C 20 60 ug mL The E coli host strain mostly influences the quality of the plasmid DNA Whereas strains like DH5a or XL1 Blue usually produce high qualit
27. ev 10 45 Plasmid DNA purification Problem Possible cause and suggestions No or low plasmid DNA yield after NucleoBond Finalizer precipitation continued Dead volume too high If high concentration of plasmid DNA is the main aim elution should be performed in small volumes Naturally a portion of the eluate will be lost in the syringe and on the NucleoBond Finalizer To minimize these losses in the second elution step try to transfer even the last droplet from the syringe to the NucleoBond Finalizer for example by tapping the NucleoBond Finalizer and syringe onto the bench top Then fill the syringe with air and press forcefully the last droplets out of the NucleoBond Finalizer Repeat this step several times You might have to practice this procedure several times to achieve optimal results An acceptable dead volume is smaller than 30 uL with NucleoBond Finalizer and 60 uL with NucleoBond Finalizer Large Elution volume too small Since there are dead volumes of about 30 uL NucleoBond Finalizer and 60 uL NucleoBond Finalizer Large reasonable elution volumes start with 200 uL NucleoBond Finalizer and 400 uL NucleoBond Finalizer Large respectively Further more smaller volumes are insufficient to wet the entire membrane and will drastically decrease your yield Refer to section 4 12 Table 4 and 5 to estimate the recovery that can be expected depending on elution buffer volume Elution
28. i Maxi kit prepare the following Dissolve the lyophilized RNase A by the addition of 1 mL of Buffer RES Wearing gloves is recommended Pipette up and down until the RNase A is dissolved completely Transfer the RNase A solution back to the bottle containing Buffer RES and shake well Note the date of RNase A addition The final concentration of RNase A is 60 ug mL Buffer RES Store Buffer RES with RNase A at 4 C The solution will be stable at this temperature for at least 6 months REF 740410 100 contains 2 x 30 mg of RNase A Make sure to dissolve RNase A of both vials each in 1 mL of Buffer RES and transfer the solution back into the bottle containing Buffer RES 24 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification 6 Safety instructions risk and safety phrases The following components of the NucleoBond Xtra kit contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section 6 1 Risk and safety phrases Component Hazard contents Hazard Risk Safety symbol phrases phrases Inhalt Gefahrstoff Gefahrstoff R S tze S S tze symbol LYS Sodium hydroxide lt 2 x Xi R 36 38 S 26 Natriumhydroxid lt 2 37 39 45 WASH Buffer salts isopropanol 10 20 v R 10 S 24 25 Puffersalze Isopropanol 10 20 ELU Buffer salts ethanol 5 20 R 10 Puffersalze Ethanol 5 20 RNase A RNase A Iyophilized x Xn R 42 43 S 22 24 RNase A lyophilisiert Ri
29. itation of salt Nucleic acid Check isopropanol purity and perform precipitation at room pellet is temperature 18 25 C but centrifuge at 4 C Do not let opaque or the eluate drip from the column into isopropanol but add white instead isopropanol to the final eluate and mix immediately of clear and Try resuspending the pellet in Buffer WASH and reload onto glassy the same NucleoBond Xtra Column Wash the column several times with Buffer WASH before loading Pellet was over dried Try to dissolve at higher temperatures for a longer period of time e g 2h at 37 C or overnight at RT preferably under constant spinning 3D shaker Nucleic Co precipitation of salt or residual alcohol acid pellet Wash the pellet again with 70 ethanol or increase the does not reconstitution buffer volume resuspend in buffer Insoluble particles in redissolved DNA Centrifuge the redissolved DNA to pellet the insoluble particles and transfer supernatant to a new tube Insoluble particles do not affect DNA quality As an alternative insoluble particles can easily be removed by using the NucleoBond Finalizer NucleoBond Xtra Midi or NucleoBond Finalizer Large NucleoBond Xtra Maxi No or low plasmid DNA yield after NucleoBond Finalizer pre cipitation Already no or low plasmid DNA after elution from the NucleoBond Xtra Column Refer to detailed troubleshooting No or low plasmid DNA yield MACHEREY NAGEL 03 2012 R
30. ke sure that plate and liquid culture contain the appropriate selective antibiotic to guarantee plasmid propagation see section 4 3 for more information Shake at 37 C and 300 rpm for 8 h a Prepare a large overnight culture Note To utilize the entire large binding capacity of the NucleoBond Xtra Columns it is important to provide enough plasmid DNA If the culture is known to grow poorly or the plasmid does not quite behave like a high copy plasmid please consult section 4 6 for larger culture volumes If you are not sure about the plasmid copy number and growth behavior of your host strain increase the culture volume and decide later in step 3 how much cells to use for the preparation The recommended culture volumes below are calculated for a final OD of around 4 see section 4 5 Inoculate an overnight culture by diluting the starter culture 1 1000 into the given volumes of LB medium also containing the appropriate selective antibiotic Grow the culture overnight at 37 C and 300 rpm for 12 16 h 100 mL 300 mL Harvest bacterial cells Measure the cell culture OD and determine the recommended culture volume V mL 400 0D V mL 1200 O0D Pellet the cells by centrifugation at 4 500 6 000 x g for 10 min at 4 C and discard the supernatant completely 28 MACHEREY NAGEL 03 2012 Rev 10 NucleoBond Xtra Midi Maxi Note It is of course possible to use la
31. kes 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 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 pa
32. lular debris into the suspension Incubate the mixture at room temperature 18 25 C for 5 min Warning Prolonged exposure to alkaline conditions can irreversibly denature and degrade plasmid DNA and liberate contaminating chromosomal DNA into the lysate Note Increase LYS buffer volume proportionally if more than the recommended cell mass is used see section 4 7 for information on optimal cell lysis MACHEREY NAGEL 03 2012 Rev 10 29 NucleoBond Xtra Midi Maxi 6 Equilibration Buffer EQU Equilibrate a NucleoBond Xtra Column together with the inserted column filter with Equilibration Buffer EQU Apply the buffer onto the rim of the column filter as shown in the picture and make sure to wet the entire filter Allow the column to empty by gravity flow The column does not run dry 7 Neutralization Buffer NEU se Add Neutralization Buffer NEU to the suspension and immediately mix the lysate gently by inverting the tube 10 15 times Do not vortex The flask or tube used for this step should not be filled more than two thirds to allow homogeneous mixing Make sure to neutralize completely to precipitate all the protein and chromosomal DNA The lysate should turn from a slimy viscous consistency to a low viscosity homogeneous suspension of an off white flocculate Immediately proceed with step 8 An incubation of the lysate is not necessary Note Increase NEU buffer volume prop
33. mL of the protocol can conveniently be used in combination with 50 mL centrifugation tubes More lysis buffer usually requires to split the sample 4 8 Difficult to Iyse strains For plasmid purification of for example Gram positive bacteria or strains with a more resistant cell wall it might be advantageous to start the preparation with a lysozyme treatment Therefore resuspend the cell pellet in Buffer RES containing 2 mg mL lysozyme and incubate at 37 C for 30 minutes Proceed then with the lysis procedure according to the NucleoBond Xtra standard protocol MACHEREY NAGEL 03 2012 Rev 10 17 Plasmid DNA purification 4 9 Setup of NucleoBond Xtra Columns Ideally the NucleoBond Xtra Midi or Maxi Columns are placed into a NucleoBond Xtra Combi Rack see ordering information They are held either by the collar ring of the cartridges or by the Plastic Washers included in the kit to individually adjust the height of each column see Figure 3 The Plastic Washers can also be used to hold the columns on top of suitable collection tubes or flasks The NucleoBond Xtra Combi Rack can be used in combination with NucleoBond PC 100 500 and 2000 as well Note that the NucleoBond Xtra Midi Columns can also be placed in the NucleoBond Rack Large REF 740563 A B Figure 3 Setup of NucleoBond Xtra Midi Maxi Columns with the NucleoBond Xtra Combi Rack A Setup for clarification loading and first washing step B
34. n of an off white floccu late Immediately proceed with step 8 of the high copy plasmid purification protocol section 7 1 An incubation of the lysate is not necessary Note Increase NEU buffer volume proportionally if more than the recommended cell mass is used see section 4 7 for information on optimal cell lysis 36 MACHEREY NAGEL 03 2012 Rev 10 NucleoBond Xtra Midi Maxi 7 3 Concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers Note Use of the NucleoBond Finalizers is only recommended for vector sizes smaller than 50 kbp Midi NucleoBond Maxi NucleoBond Finalizer Finalizer Large 1 Precipitate DNA Note Check DNA concentration photometrically before precipitation This helps to choose the best buffer volume in step 5 and allows calculation of the recovery after concentration Add 0 7 volumes of room temperature isopropanol not supplied with the kit Vortex well and let the mixture sit for 2 minutes E g for 5 mL NucleoBond Xtra Midi eluate add 3 5 mL isopropanol for 15 mL NucleoBond Xtra Maxi eluate add 10 5 mL isopropanol 3 5 mL for 10 5 mL for 5 mL eluate 15 mL eluate 2 Load precipitate Remove the plunger from a 30 mL Syringe and attach a NucleoBond Finalizer to the outlet Fill the precipitation mixture into the syringe insert the plunger hold the syringe in a vertical position and press the mixture slowly thr
35. oBond Xtra plasmid purification 7 1 High copy plasmid purification Midi Maxi 7 2 Low copy plasmid purification Midi Maxi 7 3 Concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers 11 11 11 13 14 15 16 17 17 18 19 19 20 23 23 24 25 25 26 28 28 34 37 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification 8 Appendix 8 1 Troubleshooting 8 2 Ordering information 8 3 Product use restriction warranty 40 40 48 49 4 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification 1 Components 1 1 Kit contents NucleoBond Xtra NucleoBond Xtra Midi Midi Plus 10 preps 50preps 100preps 10 preps 50 preps REF 740410 10 740410 50 740410 100 740412 10 740412 50 Buffer RES 100 mL 500 mL 1000 mL 100 mL 500 mL Buffer LYS 4x25mL 500 mL 1000 mL 4x25mL 500 mL Buffer NEU 100 mL 500 mL 1000 mL 100 mL 500 mL Buffer EQU 200 mL 2x500mL 2x1000 mL 200 mL 2x500 mL Buffer WASH 100 mL 500 mL 1000 mL 100 mL 500 mL Buffer ELU 60 mL 300 mL 600 mL 60 mL 300 mL RNase A 6 mg 30 mg 2x30 mg 6 mg 30 mg Iyophilized NucleoBond Xtra 10 50 100 10 50 Midi Columns incl NucleoBond Xtra Midi Column Filters NucleoBond 10 50 Finalizers 30 mL Syringes 10 50 1 mL Syringes 10 50 Buffer TRIS 15 mL 75 mL Plastic Washers 5 10 10 5 10 User manual 1 1 1 1 1 For preparation of working solutions and storage conditions see sec
36. ortionally if more than the recommended cell mass is used see section 4 7 for information on optimal cell lysis 30 MACHEREY NAGEL 03 2012 Rev 10 NucleoBond Xtra Midi Maxi 2 Clarification and loading Make sure to have a homogeneous suspension of the precipitate by inverting the tube 3 times directly before applying the lysate to the equilibrated NucleoBond Xtra Column Filter to avoid clogging of the filter The lysate is simultaneously cleared and loaded onto the column Refill the filter if more lysate has to be loaded than the filter is able to hold Allow the column to empty by gravity flow Alternative The precipitate can be removed by centrifugation at 5 000 x g for at least 10 min for example if more than double the recommended cell mass was used If the supernatant still contains suspended matter transfer it to a new tube and repeat the centrifugation preferably at higher speed or apply the lysate to the equilibrated NucleoBond Xtra Column Filter This clarification step is extremely important since residual precipitate may clog the NucleoBond Xtra Column To load the column you can either apply the cleared lysate to the equilibrated filter or remove the unused filter beforehand Allow the column to empty by gravity flow Note You may want to save all or part of the flow through for analysis see section 8 1 e Wash column filter and column Buffer EQU Wash
37. ough the NucleoBond Finalizer the mixture should pass the NucleoBond Finalizer drop by drop Discard the flow through 3 Wash precipitate Remove the NucleoBond Finalizer from the syringe pull out the plunger and reattach the NucleoBond Finalizer to the syringe outlet Fill 70 ethanol not supplied with the kit into the syringe insert the plunger hold the syringe in a vertical position and press the ethanol slowly through the NucleoBond Finalizer Discard the flow through 2mL 5 mL MACHEREY NAGEL 03 2012 Rev 10 37 NucleoBond Xtra Midi Maxi Midi NucleoBond Maxi NucleoBond Finalizer Finalizer Large 4 Dry filter membrane Remove the NucleoBond Finalizer from the syringe pull out the plunger and reattach the NucleoBond Finalizer Press air through the NucleoBond Finalizer as strongly as possible while touching a tissue with the tip of the NucleoBond Finalizer to soak up ethanol Repeat this step at least as often as indicated below until no more ethanol leaks from the NucleoBond Finalizer Note A new dry syringe can be used to speed up the procedure not provided gt 3 times until dry 6 times until dry Optional You can incubate the NucleoBond Finalizer for 10 minutes at 80 C to minimize ethanol carry over However the final recovery may be reduced by over drying the DNA 5 Elute DNA Buffer TRIS Remove the NucleoBond Finalizer from
38. purification 4 5 Culture volume for high copy plasmids Due to the influence of growth media TB CircleGrow 2xYT growth conditions shaking temperature host strain or type of plasmid insert etc the final amount of cells in a bacterial culture can vary over a wide range By rule of thumb 1 liter of E coli LB culture with an OD of 1 consists of 1 x 10 cells and yields about 1 5 1 8 g cell wet weight Overnight cultures grown in LB medium usually reach an OD of 3 6 under vigorous shaking in flasks Fermentation cultures even reach an OD of 10 and more The expected DNA yield for a high copy plasmid is approximately 1 mg per gram cell wet weight It is therefore important to adjust the cell mass rather than the culture volume for the best plasmid purification results But since the cell mass or cell wet weight is tedious to determine it was replaced in this manual by the mathematical product of optical density at 600 nm OD and culture volume Vol two variables that are much easier to measure 600 ODV OD x Vol mL 600 Note that for a correct OD determination the culture samples have to be diluted if OD exceeds 0 5 in order to increase proportionally with cell mass For a well grown E coli culture a 1 10 dilution with fresh culture medium is recommended The measured OD is then multiplied with the dilution factor 10 to result in a theoretical OD value This OD is used in Table 2 to determine the appropria
39. ra Buffer Set Buffer RES LYS NEU RNase A only applicable with NucleoBond Xtra kits sufficient for 12 Xtra Maxi and 18 Xtra Midi preps Buffer WASH NucleoBond Finalizer for use with NucleoBond Xtra Midi Midi EF NucleoBond PC 100 PC 500 PC 500 EF NucleoBond Finalizer Plus for use with NucleoBond Xtra Midi Midi EF NucleoBond PC 100 PC 500 PC 500 EF NucleoBond Finalizer Large for use with NucleoBond Xtra Maxi Maxi EF NucleoBond PC 2000 PC 2000 EF NucleoBond Finalizer Large Plus for use with NucleoBond Xtra Maxi Maxi EF NucleoBond PC 2000 PC 2000 EF RNase A Visit www mn net com for more detailed product information 740410 10 50 100 740412 10 50 740414 10 50 100 740416 10 50 740415 740417 740375 1000 740519 20 740520 20 740418 20 740419 20 740505 50 740505 10 50 100 preps 10 50 preps 10 50 100 preps 10 50 preps 1000 mL 20 filters 2 syringe sets 20 filters 20 syringe sets 20 large filters 2 syringe sets 20 large filters 20 syringe sets 50 mg 100 mg 48 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification 8 3 Product use restriction warranty NucleoBond Xtra Midi Maxi NucleoSpin Plasmid 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 produ
40. rger culture volumes for example if the plasmid does not behave like a typical high copy vector see section 4 6 for more information In this case increase RES LYS and NEU buffer volumes proportionally in steps 4 5 and 7 Additional lysis buffer might have to be ordered separately see ordering information for NucleoBond Xtra Buffer Set I section 8 2 If the culture volume is more than double the recommended culture volume it is advantageous to use a centrifuge for the lysate clarification in step 8 rather than the NucleoBond Xtra Column Filters o gt 4 Resuspension Buffer RES Resuspend the cell pellet completely in Resuspension Buffer RES RNase A by pipetting the cells up and down or vortexing the cells For an efficient cell lysis it is important that no clumps remain in the suspension Note Increase RES buffer volume proportionally if more than the recommended cell mass is used see section 4 7 for information on optimal cell lysis and section 4 8 regarding difficult to lyse strains 5 Cell lysis Buffer LYS Check Lysis Buffer LYS for precipitated SDS prior to use If a white precipitate is visible warm the buffer for several minutes at 30 40 C until precipitate is dissolved completely Cool buffer down to room temperature 18 25 C Add Lysis Buffer LYS to the suspension Mix gently by inverting the tube 5 times Do not vortex as this will shear and release contaminating chromosomal DNA from cel
41. rt 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 DH5a is a trademark of Life Technologies Inc NucleoSpin is a trademark of MACHEREY NAGEL GmbH amp Co KG NucleoBond is a trademark of MACHEREY NAGEL GmbH amp Co KG 50 MACHEREY NAGEL 03 2012 Rev 10
42. sk phrases R10 R 36 38 R 42 43 Flammable Entztindlich Irritating to eyes and skin Reizt die Augen und die Haut May cause sensitization by inhalation and skin contact Sensibilisierung durch Einatmen und Hautkontakt m glich Safety phrases 22 S 24 S 24 25 S 26 S 37 39 S45 Do not breathe dust Staub nicht einatmen Avoid contact with the skin Ber hrung mit der Haut vermeiden Avoid contact with the eyes and the skin Ber hrung mit den Augen und der Haut vermeiden In case of contact with eyes rinse immediately with plenty of water and seek medical advice Bei Ber hrung mit den Augen gr ndlich mit Wasser absp len und Arzt konsultieren Wear suitable protective clothing and gloves Bei der Arbeit geeignete Schutzhandschuhe und Schutzbrille Gesichtsschutz tragen In case of accident or if you feel unwell seek medical advice immediately show the label where possible Bei Unfall oder Unwohlsein sofort Arzt hinzuziehen wenn m glich dieses Etikett vorzeigen Hazard labeling not neccessary 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 03 2012 Rev 10 25 Plasmid DNA purification 6 2 GHS classification Only harmful features need not be labeled with H and P phrases
43. te culture volume Table 2 shows recommended ODVs and the corresponding pairs of OD and culture volume that can be easily handled using the standard kit protocol lysis buffer volumes For example if the OD of your E coli culture is 6 use 66 mL culture for a Midi prep or 200 mL for a Maxi prep 600 Table 2 Recommended culture volumes for high copy plasmids NucleoBond Pellet Rec OD OD OD OD OD Xtra wet ODV weight 2 4 6 8 10 Midi 0 75 g 400 200 mL 100 mL 66 mL 50 mL 40 mL Maxi 2 25 g 1200 600 mL 300 mL 200 mL 150 mL 120 mL MACHEREY NAGEL 03 2012 Rev 10 15 Plasmid DNA purification 4 6 Culture volume for low copy plasmids NucleoBond Xtra kits are designed for isolation of high copy plasmids up to several hundred copies cell as well as low copy plasmids lt 20 copies cell However when purifying low copy plasmids the cell mass and the lysis buffer volumes should be increased at least by factor 2 to provide enough DNA to utilize the columns binding capacity Table 3 shows recommended ODVs and the corresponding pairs of OD and culture volume for low copy plasmid cell cultures for detailed information on calculating ODV OD x Vol refer to section 4 5 For example if the OD of your E coli culture is 6 use 133 mL culture for a Midi prep or 400 mL for a Maxi prep Table 3 Recommended culture volumes for low copy plasmids
44. ter lane 4 purification using NucleoBond Xtra Midi are shown with a recovery of gt 90 MACHEREY NAGEL 03 2012 Rev 10 41 Plasmid DNA purification Problem Possible cause and suggestions No or low plasmid DNA yield Plasmid did not propagate Check plasmid content in the cleared lysate see Figure 6 Use colonies from fresh plates for inoculation and add fresh selective antibiotic to plates and media Estimate plasmid content prior to large purifications by a quick NucleoSpin Plasmid or NucleoSpin Plasmid QuickPure preparation Alkaline lysis was inefficient Too much cell mass was used Refer to section 4 5 4 7 regarding recommended culture volumes and lysis buffer volumes Check plasmid content in the cleared lysate see Figure 6 Check Buffer LYS for SDS precipitation before use especially after storage below 20 C If necessary incubate the bottle for several minutes at 30 40 C and mix well until SDS is redissolved SDS or other precipitates are present in the sample Load the crude lysate onto the NucleoBond Xtra Column Filter inserted in the NucleoBond Xtra Column This ensures complete removal of SDS precipitates Incubation of cleared lysates for longer periods of time might lead to formation of new precipitate If precipitate is visible it is recommended to filter and centrifuge the lysate again directly before loading it onto the NucleoBond Xtra Column Sample lysate is
45. the syringe pull out the plunger of a 1 mL Syringe and attach the NucleoBond Finalizer to the syringe outlet Note Refer to section 4 12 Table 4 Midi or 5 Maxi to choose the appropriate volume of elution buffer Pipette an appropriate volume of Redissolving Buffer TRIS 5 mM Tris HCl pH 8 5 or TE buffer into the syringe see section 4 12 Do not use pure water unless pH is definitely higher than 7 0 Place the NucleoBond Finalizer outlet in a vertical position over a fresh collection tube not provided and elute plasmid DNA very slowly drop by drop by inserting the plunger 200 800 uL 400 1000 pL Remove the NucleoBond Finalizer from the syringe pull out the plunger and reattach the NucleoBond Finalizer to the syringe outlet Transfer the first eluate back into the syringe and elute into the same collection tube a second time Load first eluate Load first eluate completely completely 38 MACHEREY NAGEL 03 2012 Rev 10 NucleoBond Xtra Midi Maxi Midi NucleoBond Maxi NucleoBond Finalizer Finalizer Large Remove the NucleoBond Finalizer from the syringe pull out the plunger to aspirate air reattach the NucleoBond Finalizer and press the air out again to force out as much eluate as possible Determine plasmid yield by UV spectroscopy and confirm plasmid integrity by agarose gel electrophoresis see section 4 13 MACHEREY NAGEL 03 2012 Rev 10 39
46. ting and dead volume a minimal amount of DNA has to be loaded to achieve a desired concentration If possible try to pool several DNA precipitation batches since percentage of recovery and concentration significantly increase with higher amounts of loaded DNA Purified plasmid does not perform well in subsequent reactions Plasmid DNA is contaminated with chromosomal DNA or RNA Refer to the detailed troubleshooting above Plasmid DNA is contaminated with residual alcohol Plasmid DNA was not dried completely before redissolving Precipitate DNA again by adding 1 10 volume of 3 M NaAc pH 5 0 and 0 7 volumes of isopropanol Proceed with the precipitation protocol in this manual and dry DNA pellet completely DNA is degraded Make sure that your entire equipment pipettes centrifuge tubes etc is clean and nuclease free Do not Iyse the sample with Buffer LYS for more than 5 min DNA is irreversibly denatured A denatured plasmid band runs faster on the gel than the supercoiled conformation Do not lyse the sample after addition of Buffer LYS for more than 5 minutes MACHEREY NAGEL 03 2012 Rev 10 47 Plasmid DNA purification 8 2 Ordering information Product REF Pack of NucleoBond Xtra Midi NucleoBond Xtra Midi Plus incl NucleoBond Finalizers NucleoBond Xtra Maxi NucleoBond Xtra Maxi Plus incl NucleoBond Finalizers Large NucleoBond Xtra Combi Rack NucleoBond Xt
47. tion 5 MACHEREY NAGEL 03 2012 Rev 10 5 Plasmid DNA purification 1 1 Kit contents continued NucleoBond Xtra NucleoBond Xtra NEST Maxi Plus 10 preps 50preps 100 preps 10 preps 50 preps REF 740414 10 740414 50 740414 100 740416 10 740416 50 Buffer RES 150 mL 750 mL 2 x 750 mL 150 mL 750 mL Buffer LYS 150 mL 750 mL 2x 750m 150 mL 750 mL Buffer NEU 150 mL 750 mL 2 x 750 mL 150 mL 750 mL Buffer EQU 2x 250 mL 2x 1000 mL 5x 1000 mL 2x 250 mL 2x 1000 mL 500 mL 500 mL Buffer WASH 2x150mL 1000 mL 3x1000mL 2x150mL 1000 mL 500 mL 500 mL Buffer ELU 180 mL 900 mL 2 x 900 mL 180 mL 900 mL RNase A 10 mg 50 mg 2x 50 mg 10 mg 50 mg lyophilized NucleoBond 10 50 100 10 50 Xtra Maxi Columns incl NucleoBond Xtra Maxi Column Filters NucleoBond 10 50 Finalizers Large 30 mL Syringes 10 50 1 mL Syringes 10 50 Buffer TRIS 15 mL 75 mL Plastic Washers 5 10 10 5 10 User manual 1 1 1 1 1 For preparation of working solutions and storage conditions see section 5 6 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification 1 2 Reagents and equipment to be supplied by user Reagents Isopropanol room temperatured 70 ethanol room temperatured Buffer for reconstitution of DNA for example TE buffer or sterile H O not necessary for NucleoBond Xtra Midi Maxi Plus kits Equipment Standard microbiological equipment for growing and harvesting bacteria e g
48. tion or sequencing reactions All NucleoBond Xtra eluates already contain enough salt for an isopropanol precipitation of DNA Therefore the precipitation is started by directly adding 0 7 volumes of isopropanol To prevent co precipitation of salt use room temperature 18 25 C isopropanol only and do not let the plasmid DNA solution drop into a vial with isopropanol but add isopropanol to the final eluate and mix immediately Afterwards either follow the centrifugation protocol given after the NucleoBond Xtra purification protocol or follow the support protocol for the NucleoBond Finalizers in section 7 3 to eliminate the time consuming centrifugation steps for precipitation use of NucleoBond Finalizers is only recommended for vector sizes smaller than 50 kbp The NucleoBond Finalizers are designed for quick concentration and desalination of plasmid and cosmid DNA eluates that are obtained by anion exchange chromatography based DNA purification The sample is precipitated with isopropanol as mentioned above and loaded onto a special silica membrane by means of a syringe After an ethanolic washing step the membrane is dried by pressing air through the filter Elution of pure DNA is carried out with slightly alkaline low salt buffers like Buffer TRIS 5 mM Tris HCl pH 8 5 provided with all NucleoBond Xtra Plus kits or TE buffer 10 mM Tris HCl pH 7 5 1 mM EDTA Do not use pure water unless pH is definitely higher than 7 0 For ma
49. to the suspension Note Increase LYS buffer volume proportionally if more than the recommended cell mass is used see section 4 7 for information on optimal cell lysis Mix gently by inverting the tube 5 times Do not vortex as this will shear and release contaminating chromosomal DNA from cellular debris into the suspension Incubate the mixture at room temperature 18 25 C for 5 min Warning Prolonged exposure to alkaline conditions can irreversibly denature and degrade plasmid DNA and liberate contaminating chromosomal DNA into the lysate MACHEREY NAGEL 03 2012 Rev 10 35 NucleoBond Xtra Midi Maxi 6 Equilibration Buffer EQU Equilibrate a NucleoBond Xtra Column together with the inserted column filter with Equilibration Buffer EQU Apply the buffer onto the rim of the column filter as shown in the picture and make sure to wet the entire filter Allow the column to empty by gravity flow The column does not run dry 7 Neutralization Buffer NEU Add Neutralization Buffer NEU to the suspension and immediately mix the lysate gently by inverting the tube 10 15 times Do not vortex The flask or tube used for this step should not be filled more than two thirds to allow homogeneous mixing Make sure to neutralize completely to precipitate all the protein and chromosomal DNA The lysate should turn from a slimy viscous consistency to a low viscosity homogeneous suspensio
50. too fast Plasmid DNA needs time to dissolve Elute really very slowly drop by drop Repeat the elution procedure using the first eluate Forgot to elute a second time Repeating the elution procedure with the first eluate is crucial for optimal yields However eluting a third time shows no further improvement Plasmid size Precipitation efficiency is almost independent of plasmid size but elution from the NucleoBond Finalizers becomes more and more difficult with increasing size of the construct If you face low yields with large cosmids you may try heating the NucleoBond Finalizer the syringes and elution buffer to 70 C 46 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification Problem Possible cause and suggestions Low overall yield Refer to detailed troubleshooting No or low plasmid DNA yield and lower your elution buffer volume Refer to section 4 12 Table 4 and 5 to estimate the DNA concentrations that can be expected Fresh elution buffer was used for second elution step Low DNA The second elution step is crucial for optimal yield but to concentration achieve a high DNA concentration the eluate of the first elution after step has to be used for the second elution NucleoBond Finalizer precipitation Not enough DNA loaded Since there is a technical limitation to at least 200 uL NucleoBond Finalizer and 400 uL NucleoBond Finalizer Large of elution buffer due to membrane wet
51. too viscous Too much cell mass was used Refer to section 4 5 4 7 regarding recommended culture volumes and lysis buffer volumes Make sure to mix well after neutralization to completely precipitate SDS and chromosomal DNA Otherwise filtration efficiency and flow rate go down and SDS prevents DNA from binding to the column PH or salt concentrations of buffers are too high Check plasmid content in the wash fractions see Figure 6 Keep all buffers tightly closed Check and adjust pH of Buffer EQU pH 6 5 WASH pH 7 0 and ELU pH 9 0 with HCI or NaOH if necessary 42 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification Problem Possible cause and suggestions Culture volumes are too large Refer to section 4 5 4 7 regarding recommended culture volumes and larger Iysis buffer volumes NucleoBond an Precipitate was not resuspended before loading l 3 k P during filtra Invert crude lysate at least 3 times directly before loading tion Incomplete precipitation step Make sure to mix well after neutralization to completely precipitate SDS and chromosomal DNA NucleoBond Sample is too viscous Do NOT attempt to purify lysate prepared from a culture volume larger than recommended for any given column size with standard lysis buffer volumes Incomplete lysis not only blocks the column but can also significantly reduce yields Refer to section 4 5 and 4 6 for recommended culture volumes and section 4
52. tra buffer volumes standard protocol are adjusted to ensure optimal lysis for culture volumes appropriate for high copy plasmids according to section 4 5 Table 2 Using too much cell material leads to inefficient cell lysis and precipitation and might reduce plasmid yield and purity Therefore lysis buffer volumes should be increased when applying larger culture volumes in case of for example low copy vector purification section 4 6 Table 3 By rule of thumb calculate the necessary lysis buffer volumes for RES LYS and NEU as follows Vol mL Culture Volume mL x OD 50 For example if 200 mL of a low copy bacterial culture OD 4 is to be lysed the appropriate volumes of lysis buffers RES LYS and NEU are 16 mL each If more lysis buffer is needed than is provided with the kit an additional buffer set including buffers RES LYS NEU and RNase A can be ordered separately see ordering information By using sufficient amounts of lysis buffer lysis time can be limited to 3 4 minutes and should not exceed 5 minutes Prolonged exposure to alkaline conditions can irreversibly denature and degrade plasmid DNA and liberate contaminating chromosomal DNA into the lysate Please note that the calculated lysis buffer volumes for NucleoBond Xtra preparations do not match the recommended volumes in the protocol due to the fact that most users start with much less cells than the recommended ODV 1200 Furthermore the 2 x 12
53. ty can be checked by running the precipitated samples on a 1 agarose gel This will give information on conformation and structural integrity of isolated plasmid DNA i e it shows whether the sample is predominantly present in the favorable super coiled form ccc usually the fastest band as an open circle oc or even in a linear form see section 8 1 Figure 6 4 14 Convenient stopping points Cell pellets can easily be stored for several months at 20 C Cleared lysates can be kept on ice or at 4 C for several days For optimal performance the column purification should not be interrupted However the columns can be left unattended for several hours since the columns do not run dry This might cause only small losses in DNA yield The eluate can be stored for several days at 4 C Note that the eluate should be warmed up to room temperature before precipitating the DNA to avoid co precipitation of salt MACHEREY NAGEL 03 2012 Rev 10 23 Plasmid DNA purification 5 Storage conditions and preparation of working solutions All kit components can be stored at room temperature 18 25 C and are stable at least two years Storage of Buffer LYS 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 Xtra Mid
54. uestions 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 lysate and the lysate flow through samples lanes 1 and 2 It might also occur in the wash fraction but must be absent in the eluate 40 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification Genomic DNA should not be visible at all but would show up in the gel slot or right below indicating for example too harsh Iysis conditions M 1 u eet dez 4 5 Marker 2 Hindlll cleared lysate ccc linear and oc structure of the plasmid degraded RNA Il lysate flow through no plasmid DNA but degraded RNA lI wash flow through no plasmid DNA or residual RNA IV eluate pure plasmid DNA EcoRI restriction linearized form of plasmid Figure 6 Exemplary analytical check of NucleoBond Xtra Midi purification samples Plasmid pUC18 bacterial strain E coliDH5 20 uL of each precipitated sample has been analyzed on a 1 agarose gel Equal amounts of plasmid DNA before lane 1 and af
55. unken offener Flamme hei en Oberfl chen fernhalten Nicht rauchen P 233 Keep container tightly closed Beh lter dicht verschlossen halten P 234 Keep only in original container Nur im Originalbeh lter aufbewahren 26 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification Precaution phrases 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 Avoid breathing dust Einatmen von Staub vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF ON SKIN Wash with plenty of soap and water BEI KONTAKT MIT DER HAUT Mit viel Wasser und Seife 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 minutes 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
56. 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 Sodium hydroxide lt 2 Warning 290 315 234 280 Natriumhydroxid lt 2 Achtung 319 302 352 305 351 338 332 313 337 313 390 406 WASH Buffer salts isopropanol Warning 226 210 233 10 20 403 235 Puffersalze Isopropanol Achtung 10 20 ELU Buffer salts ethanol Warning 226 319 210 233 280 5 20 305 351 338 Puffersalze Ethanol 5 20 Achtung 337 313 403 235 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 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 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 verursachen Precaution phrases P 210 Keep away from heat sparks open flames hot surfaces No smoking Von Hitze F
57. urity Argo Asso lt 1 8 No nucleic acid pellet formed after precipitation NucleoBond Xtra Column Filter was not removed before second washing step Protein content too high due to inefficient washing Remove the NucleoBond Xtra Column Filter before performing the second washing step with Buffer WASH Buffer WASH was used instead of Buffer EQU for the first wash Buffer EQU has to be used to wash out the NucleoBond Xtra Column Filter to avoid SDS carry over Only minimal amounts of DNA were loaded onto the column Excess free binding capacity requires more extensive washing double washing step with Buffer WASH Reduce lysis time lt 5 min Pellet was lost Handle the precipitate with care Decant solutions carefully Determine DNA yield in Buffer ELU in order to calculate the amount of plasmid DNA that should be recovered after precipitation Plasmid DNA might be smeared over the wall of the tube Dissolve DNA with an appropriate volume of reconstitution buffer by rolling the tube for at least 30 min 44 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification Problem Possible cause and suggestions No nucleic Nucleic acid did not precipitate acid pellet Check type and volumes of precipitating solvent Make sure formed after precipitation to use at least 0 7 volumes of isopropanol and mix thoroughly Centrifuge for longer periods of time at higher speed continued Co precip
58. ximum yield it is recommended to perform the elution step twice The first elution step is done using fresh buffer whereas in the second elution step the eluate from the first elution is reapplied on the NucleoBond Finalizer to allow complete solubilization of the plasmid DNA recovery highly depends on the used elution buffer volume Large volumes result in a high recovery of up to 90 but in a lower DNA concentration Small elution volumes on the other hand increase the concentration but at the cost of DNA yield If a small volume is chosen make sure to recover as much eluate as possible from the syringe and NucleoBond Finalizer by pressing air through the NucleoBond Finalizer several times after elution and collecting every single droplet to minimize the dead volume Figure 4 and 5 illustrate exemplarily how DNA recovery and final DNA concentration depend on the buffer volume which is used for elution of DNA from the NucleoBond Finalizer and NucleoBond Finalizer Large respectively 20 MACHEREY NAGEL 03 2012 Rev 10 Plasmid DNA purification 100 2 0 90 r1 8 80 71 6 2 X 70 r1 4 ey 60 r1 2 5 Recovery 3 50 1 0 w Concentration 3 40 0 8 o 30 r0 6 20 r0 4 Oo 10 0 2 0 T T T T T 0 0 0 200 400 600 800 1000 Elution volume uL Figure 4 Final DNA recovery and concentration after NucleoBond Finalizer application A NucleoBond Xtra Midi eluate containing 250 ug plasmid
59. y super coiled plasmid DNA other strains like for example HB101 with high levels of endonuclease activity might yield lower quality plasmid giving poor results in downstream applications like enzymatic restriction or sequencing Maniatis T Fritsch EF Sambrook J Molecular cloning A laboratory manual Cold Spring Harbor Cold Spring New York 1982 MACHEREY NAGEL 03 2012 Rev 10 13 Plasmid DNA purification The type of plasmid especially the size and the origin of replication ori has a crucial influence on DNA yield In general the larger the plasmid or the cloned insert is the lower is the expected DNA yield due to a lower copy number Even a high copy construct based on a ColE1 ori can behave like a low copy vector in case of a large or unfavorable insert In addition the ori itself influences the yield by factor 10 100 Thus plasmids based on for example pBR322 or pACYC cosmids or BACs are maintained at copy numbers lt 20 down to even only 1 whereas vectors based on for example 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 has to be achieved Culture volume and lysis procedures have to be adjusted accordingly 4 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 ph

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