Endotoxin-free plasmid DNA purification User manual
Contents
1. Add Buffer S2 EF to the suspension Mix gently by inverting the tube 6 8 times Incubate the mixture at room temperature 18 25 C for 2 3 min max 5 min Do not vortex as this will release contaminating chromosomal DNA from cellular debris into the suspension 12 mL 40 mL 120 mL Add pre cooled Buffer S3 EF 4 C to the suspension Immediately mix the lysate gently by inverting the flask 6 8 times until a homogeneous suspension containing an off white flocculate is formed 12 mL 40 mL 120 mL 24 MACHEREY NAGEL 03 2014 Rev 13 NucleoBond PC EF AX 500 AX 2000 AX 10000 AX 500 Maxi Incubate the suspension for 5min on ice before continuing with step 4 Clarification of the lysate AX 2000 10000 Mega Giga Pour the lysate immediately into the NucleoBond Bottle Top Filter Type 1 AX 2000 or Type 2 AX 10000 and continue as described in step 4 of this protocol To save time the equilibration of the NucleoBond Columns see step 3 can be started during the clarification of the lysate as described in step 4 Equilibration of the column Equilibrate a NucleoBond Column with Buffer N2 EF Allow the column to empty by gravity flow Discard flow through 25 mL 100 mL Clarification of the lysate Clear the bacterial lysate by using NucleoBond Folded Filters PC 500 EF or NucleoBond Bottle Top Filters PC 2000 10000 EF2. Endotoxin free plasmid DNA purification User manual NucleoBond PC 500 EF NucleoBond PC 2000 EF NucleoBond PC 10000 EF NucleoBond PC Prep 100 March 2014 Rev 13 MACHEREY NAGEL MN www mn net com Endotoxin free plasmid DNA purification Maxi Mega Giga Preparative scale Protocol at a glance Rev 13 30 min at 12 C 30 min at 12 C 30 min at 12 C Maxi Mega Giga Preparative scale AX 500 AX 2000 AX 10000 AX Prep 100 1 nn of 4 500 6 000xg 4 500 6 000xg 4 500 6 000 x g es 4 500 6 000 x g es bacterial cells 15 min at 4 C 15 min at 4 C 15 min at 4 C 15 min at 4 C 2 Cells lysis Buffer S1 EF 12 mL 40 mL 120 mL 1000 mL Buffer S2 EF 12 mL 40 mL 120 mL 1000 mL lt 5 min at RT lt 5 min at RT lt 5 min at RT lt 3 min at RT Buffer S3 EF 12 mL 40 mL 120 mL gt 1000 mL 5 min at 0 C 25 min at 0 C Mix thoroughly invert 10 15 times 3 Equilibration of the column Butfer N2 EF Buffer N2 EF Buffer N2 EF Buffer N2 EF 5 mL 25 mL 100 mL 900 mL 4 Clarification of the lysate Folded Filter Bottle Top Filter Bottle Top Filter 1 Sieving Fabric Type 1 Type 2 j i j 2nd Folded Filters 20 min 5 min 5 min 5 Binding lt Load cleared Load cleared Load cleared Load cleared lysate onto
3. This step is extremely important excess precipitate left in suspension may clog the NucleoBond Column in later steps AX 500 Maxi Place the NucleoBond Folded Filter in a small funnel for support and pre wet the filter with a few drops of Buffer N2 EF or sterile deionized H O Load the bacterial lysate onto the wet filter and collect the flow through AX 2000 10000 Mega Giga Pour the lysate immediately into the NucleoBond Bottle Top Filter Type 1 AX 2000 or Type 2 AX 10000 and incubate at room temperature for 10 min Switch on the vacuum source optimal 0 4 to 0 6 bar in order to filtrate the lysate through the NucleoBond Bottle Top Filter After all liquid has passed the filter 3 5 min switch off the vacuum source AX 10000 Giga It is possible to stir the precipitate with a spatula gently onto the filter In order to recover residual liquid switch on vacuum again for another minute 20 min 5 min smin Alternatively Centrifuge the crude lysate at high speed gt 4 5 12 000 x g at 4 C for 40 min Maxi 50 min Mega and 60 min Giga Subsequently after centrifugation carefully remove the supernatant from the white precipitate and load it onto the equilibrated NucleoBond Column MACHEREY NAGEL 03 2014 Rev 13 25 NucleoBond PC EF AX 500 AX 2000 AX 10000 5 Binding Load the cleared Iysate from step 4 onto the Nucleo
4. 180 min 20 h 2 preps 2 preps 2 preps prep NucleoBond PC EF Prep 100 kits allow the purification of DNA that fulfills the following criteria Table 2 Criteria Parameter Method Criterion Structural integrity Agarose gel gt 90 ccc Photo documentation RNA ssDNA HPLC lt 50 ug mg Agarose gel Chrom DNA HPLC lt 50 ug mg Southern Blot PCR Endotoxin LAL test lt 0 1 EU ug Protein Bradford lt 10 ug mg Purity A eo A80 1 80 1 95 UV spectrum 220 320 nm normal MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification All NucleoBond Columns are resistant to organic solvents such as alcohol chloroform and phenol and are free of DNase and RNase NucleoBond AX Resin can be used over a wide pH range from pH 2 5 8 5 and can remain in contact with buffers for up to three hours without any change in its chromatographic properties After three hours nucleic acids will begin to elute at increasingly lower salt concentrations Normally the resin remains functional in buffers containing up to 2 M salt It remains intact in the presence of denaturing agents like formamide urea or common detergents such as Triton X 100 and NP 40 MACHEREY NAGEL 03 2014 Rev 13 9 Endotoxin free plasmid DNA purification 3 About this user manual Experienced users who are performing the purification of high copy plasmids using a NucleoBond PC EF purification kit may refer to the
5. arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL 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 mai
6. bottle top filter to a suitable flask e g Schott load the bacterial lysate and apply the vacuum After 5 min the solution will have passed through Load the resulting clear lysate onto the corresponding NucleoBond AX Column and discard the bottle top filter Figure 1 Correct use of the NucleoBond Bottle Top Filter For the NucleoBond AX 500 Column use the supplied NucleoBond Folded Filters for filtration of the lysate Figure 2 Folded filters are designed to eliminate the centrifugation step after alkaline lysis for plasmid isolation The filters completely remove SDS and precipitate cellular debris from plasmid samples For correct use please follow the instructions given in step 4 of the corresponding protocol Figure 2 Correct use of the NucleoBond Folded Filter NucleoBond Column placed in a Plastic Washer MACHEREY NAGEL 03 2014 Rev 13 13 Endotoxin free plasmid DNA purification 5 4 Analytical check refers only to PC Prep 100 Before starting the NucleoBond PC Prep 100 purification we recommend checking the fermented cell material by purifying the plasmid DNA from 20 mL and 40 mL of culture on a NucleoBond AX 100 Column supplied Follow the instructions of the attached protocol From the yield resulting from 20 mL and 40 mL fermentation broth using a NucleoBond AX 100 Column the total yield of the NucleoBond Prep 100 procedure can be estimated If necessary the culture volume
7. can be adapted accordingly with respect to a maximum pellet weight of 90 g and a maximum binding capacity of 100 mg also see section 3 2 In the case that a 20 mL fermentation broth high copy plasmid already results in 60 100 ug of plasmid DNA yields obtained from a 40 mL fermentation broth will be invalid because the NucleoBond AX 100 Column Midi prep has been overloaded Take the yields obtained from the 20 mL fermentation broth as a basis for the calculation of how much fermentation broth can be used for the NucleoBond AX Prep 100 Column preparative scale Midi AX 100 1 Harvest of bacteria Harvest bacteria from 20 and 40 mL fermentation broth by centrifugation at 4 500 6 000 x g for 15 min at 4 C 2 Cell lysis Carefully resuspend the pellet of bacterial cells in Buffer S1 EF RNase A Please see section 6 3 regarding difficult to lyse strains 4mL Add Buffer S2 EF to the suspension Mix gently by inverting the tube 6 8 times Incubate the mixture at room temperature 18 25 C for 2 3 min max 5 min Do not vortex as this will release contaminating chromosomal DNA from cellular debris into the suspension 4 mL 14 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification Midi AX 100 Add precooled Buffer S3 EF 4 C to the suspension Immediately mix the lysate gently by inverting the flask 6 8 times until a homogeneous suspension containing an off white floccu
8. chromosomal DNA At least for Mega Giga and preparative scale PC Prep 100 preps the use of an ap propriate fermentation system is recommended in order to optimize cultivation conditions 8 3 Difficult to Iyse strains Isolate plasmid DNA from difficult to lyse strains by first resuspending the pellet in Buffer S1 EF containing lysozyme 2 mg mL final concentration Incubate at 37 C for 30 minutes then continue with the addition of Buffer S2 EF and proceed with the appropriate NucleoBond protocol 22 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification 8 4 Chloramphenicol amplification of low copy plasmids To dramatically increase the low copy number of pMB1 colE1 derived plasmids grow the cell culture to mid or late log phase OD 0 6 2 0 under selective conditions with an appropriate antibiotic Then add 170 ug mL chloramphenicol and continue incubation for a further 8 12 hours Chloramphenicol inhibits host protein synthesis and thus prevents replication of the host chromosome Plasmid replication however is independent of newly synthesized proteins and continues for several hours until up to 2000 3000 copies per cell are accumulated Alternatively the cell culture can be grown with only partial inhibition of protein synthesis under low chloramphenicol concentrations 10 20 ug mL resulting in a 5 10 fold greater yield of plasmid DNA Both methods show the positive side effect of much l
9. 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 PC reduce endotoxins to a level of gt 1 EU ug However since this may be still too high for successful transfection of very sensitive cells like primary or neuronal cells NucleoBond PC EF was developed to reduce the endotoxin level to lt 0 1 EU ug plasmid DNA using a patented procedure MACHEREY NAGEL 03 2014 Rev 13 11 Endotoxin free plasmid DNA purification 5 NuceoBond PC EF purification system 5 1 The basic principle NucleoBond EF kits employ a modified alkaline SDS lysis procedure to prepare the bacterial cell pellet for plasmid purification Both chromosomal and plasmid DNA are denatured under these alkaline conditions Potassium acetate is then added to the denatured lysate which causes the formation of a precipitate containing chromosomal DNA and other cellular compounds The potassium acetate buffer also neutralizes the lysate Plasmid DNA which remains in solution can revert to its native supercoiled structure After equilibrating the appropriate NucleoBond Column with equil
10. rate of 15 20 mL min at room temperature First cut the tube at the column in the middle The resulting inlet tube of the column is connected to the silicon tube of a peristaltic or piston pump for loading Check the tightness of all fitting connections and use cable binders to prevent leakage where necessary If the column runs dry during use rehydrate column bed by pushing new buffer through the column upwards via a peristaltic pump 900 mL Clarification of the lysate Step I Put the Sieving Fabric 4 layers into a funnel on a 3 L Erlenmeyer flask or a 3 L glass bottle and load the lysate Collect the flow through on ice Alternatively Clarification can also be achieved by centrifuging the crude lysate at 3 000 5 000 xg for 1h at 4 C Subsequently after centrifugation remove the supernatant from the white precipitate carefully Step Il Moisten the NucleoBond Folded Filter Type 1 fixed in a funnel carefully with deionized water apply the flow through from step and collect the filtrate Apply the filtrate onto a pre wet NucleoBond Folded Filter Type 2 Collect the clear flow through and estimate the volume for step 4 Store the cleared lysate on ice Cleared lysate can be stored on ice for hours If further precipitate appears filter the lysate again before loading it onto the NucleoBond AX Prep 100 Column additional filters not included see ordering information We recommend using a 0 2 0 4 um CA filter f
11. A solution back to the bottle containing Buffer S1 EF and shake well Note the date of RNase A addition The final concentration of RNase A is 100 ug mL Buffer S1 EF Store Buffer S1 EF with RNase A at 4 C The solution will be stable at this temperature for at least 12 months Pre cool Neutralization Buffer S3 EF to 4 C The NucleoBond Bottle Top Filters are designed to be used with a 1 liter 45 mm neck vacuum resistant glass bottle bottles are not included in the kit Use any vacuum source for example a vacuum pump or house vacuum that generates reduced pressure between 0 2 and 0 8 bar Do not use scratched bottles and wear goggles when working with the NucleoBond Bottle Top Filter system under vacuum conditions Add indicated volume of 96 100 ethanol to the bottle labeled H O EF for 70 ethanol NucleoBond PC 10000 EF 5 preps 740548 NucleoBond PC 2000 EF 5 preps 740549 NucleoBond PC 500 EF 10 preps 740550 70 EtOH Concentrate 35 mL 35 mL 35 mL Add 80 mL ethanol Add 80 mL ethanol Add 80 mL ethanol 18 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification NucleoBond PC Prep 100 1 prep REF 740594 70 EtOH 2x35 mL Concentrate Add 80 mL ethanol to each bottle MACHEREY NAGEL 03 2014 Rev 13 19 Endotoxin free plasmid DNA purification 7 Safety instructions The following components of the NucleoBond PC EF and NucleoB
12. Bond Column Allow the column to empty by gravity flow Optional You may want to save all or part of the flow through for analysis 6 Washing Wash the column with Buffer N3 EF Repeat as indicated Discard flow through 2x 24mL 1x60 mL 4x 150 mL 2 x 40 mL Wash the column with Buffer N4 EF Repeat as indicated Discard flow through 2x12 mL 1x60 mL 3 x 130 mL 7 Elution Elute the plasmid DNA with Buffer N5 EF We recommend precipitating the eluate as soon as possible step 8 Nevertheless the eluate can be stored in closed vials on ice for several hours In this case the eluate should be preheated to room temperature before the plasmid DNA is precipitated 15 mL 25 mL 100 mL Optional Determine plasmid yield by UV spectrophotometry in order to adjust the desired concentration of DNA step 10 8 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Mix carefully and centrifuge at 4 5 15 000 x g for 30 min at 12 C Carefully discard the supernatant 26 MACHEREY NAGEL 03 2014 Rev 13 NucleoBond PC EF AX 500 AX 2000 AX 10000 Washing and drying Add room temperature endotoxin free 70 EtOH to the pellet Vortex briefly and centrifuge at 4 5 15 000 x g for 10 min at room temperature 18 25 C For preparation of endotoxin free 70 EtOH refer to section 4 Carefully remove ethanol from the tube with a pipette tip Allow the pellet to dry at room tempera
13. F We recommend precipitating the eluate as soon as possible step 8 Nevertheless the eluate can be stored in closed vials on ice for several hours In this case the eluate should be prewarmed to room temperature before the plasmid DNA is precipitated 5 mL Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Mix carefully and centrifuge at 15 000 x g for 30 min at 12 C Carefully discard the supernatant 3 5 mL Washing and drying Add room temperature endotoxin free 70 EtOH to the pellet Vortex briefly and centrifuge at 4 5 15 000 x g for 10 min at room temperature 18 25 C For preparation of endotoxin free 70 EtOH refer to section 4 2 mL Carefully remove ethanol from the tube with a pipette tip Allow the pellet to dry at room temperature 18 25 C at least for the indicated time Drying for longer periods will not harm the quality of the plasmid DNA but over drying may render the DNA less soluble 5 10 min 16 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification Midi AX 100 10 Reconstitution Dissolve pellet in an appropriate volume of Buffer TE EF or H O EF Depending on the type of centrifugation tube dissolve under constant spinning in a sufficient amount of buffer for 10 60 min 3D shaker Determine plasmid yield by UV spectrophotometry Confirm plasmid integrity by agarose gel electrophoresis 5 5 Elut
14. Protocol at a glance instead of this user manual The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure First time users are strongly advised to read this user manual Each procedural step in the protocols of this manual is arranged like the following example AX 500 AX 2000 AX 10000 1 Carefully resuspend the pellet of bacterial cells in Buffer S1 EF RNase A Please see section 6 3 regarding difficult to lyse strains 12 mL 40 mL 120 mL For NucleoBond PC 500 EF preparations refer to the buffer volumes and incubation times given in the white boxes For Mega and Giga preparations refer to the grey and black boxes respectively The name of the buffer is highlighted in bold type For example in a NucleoBond PC 500 EF preparation you are requested to resuspend the pelleted cells in 12 mL of Buffer S1 EF 10 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification 4 Endotoxins 4 1 Localization molecular structure and function of endotoxins In contrast to Gram positive bacteria which have only one lipid bilayer membrane surrounded by 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 en
15. biotics the bacterial host plasmid type size or copy number Therefore these factors should be taken into consideration For cultivation of bacterial cells we recommend LB medium The suggested bacterial culture volumes for each column size as well as expected plasmid yields are listed in Table 1 section 3 2 Overnight cultures in flasks usually reach under vigorous shaking an OD of 3 6 while fermentation cultures reach 10 and more Therefore please refer not only to the culture volume but also check OD and pellet wet weight particularly if richer culture media like 2x YT or TB are used If too much bacterial material is used lysis and precipitation steps are inefficient and cause decreased yield and plasmid quality As a general rule 1 liter E coli culture grown in LB medium yields a pellet of about 3 20 g wet weight The expected yield for a high copy number plasmid is 1 3 mg per gram wet weight 8 2 Selection of culture media The cultivation of cells is recommended at 37 C in LB Luria Bertani medium at constant shaking 200 250 rpm Alternatively rich media like 2x YT Yeast Tryptone or TB Terrific Broth can be used By using 2xYT or TB bacteria grow faster and reach the stationary phase much earlier than in LB medium lt 12 h This may lead to a higher percentage of dead or starving cells when starting the preparation The resulting plasmid DNA from overgrown cultures may be partially degraded or contaminated with
16. caution phrases P 210 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze hei en Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen 20 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification Precaution phrases P 233 P 234 P 261 P 280 P 302 352 P 304 340 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 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 Remove victim to fresh air and keep at rest in a position comfort able for breathing Bei Einatmen An die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert IF IN EYES Rinse continuously with water for several minutes Remove 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 ein
17. dotoxins 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 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 4 3 Removal of endotoxins Endotoxins are released from cells in small amounts during 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
18. e before the plasmid DNA is precipitated If possible check plasmid concentration at A during elution continuously to get maximal yield in minimal elution volume Store the eluate on ice during elution if possible 470 600 mL 30 MACHEREY NAGEL 03 2014 Rev 13 NucleoBond PC Prep 100 Preparative scale AX Prep 100 Optional Determine plasmid yield by UV spectrophotometry in order to adjust the desired concentration of DNA step 10 8 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Mix carefully and centrifuge at 4 5 15 000 x g for 30 min at 12 C Carefully discard the supernatant Make sure the temperature in the plasmid suspension will not exceed 25 C to prevent reduced yield In order to prevent salt precipitation temperatures below 5 C should be avoided 0 7 vol 9 Washing and drying Add room temperature endotoxin free 70 EtOH to the pellet Vortex briefly and centrifuge at 4 5 15 000 x g for 20 min at room temperature 18 25 C Repeat this step once For preparation of endotoxin free 70 EtOH refer to section 4 25 mL Carefully remove ethanol from the tube with a pipette tip Allow the pellet to dry at room temperature for at least the indicated time Drying for longer periods will not harm the quality of the plasmid DNA but over drying may render the DNA less soluble 60 min 10 Reconstitution Dissolve pellet in an appropriate volume of Buffer TE EF o
19. eck cleared lysate for precipitates especially if the lysate was stored for a longer time before loading If necessary clear the lysate again by filtration Column is blocked Lysate was not completely cleared Use additional NucleoBond Folded Filters to clear the lysate High back pressure during purification NucleoBond PC Prep Prep 100 Cleared lysate contains particulate matter Make sure that there is no cell debris in the lysate Particulate matter may clog the inlet frit or in line filters If necessary repeat filtration or centrifugation steps Be sure to equilibrate and use the column in the correct direction see protocol and scheme Lysis treatment was too harsh Be sure not to incubate the lysate in Buffer S2 EF for more than 5 min Cellular DNA Overzealous mixing during lysis allowed genomic DNA to shear off into the lysis buffer or RNA con a a tamination of ee viscous to mix properly or gently reduce plasmid DNA RNase digestion was inefficient RNase was not added to Buffer S1 EF or stored too long Add new RNase to Buffer S1 EF See ordering information section 8 3 36 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification No nucleic acid pellet formed after precipitation Pellet was lost Handle the precipitate with care Decant solutions carefully Measure DNA yield in Buffer N5 EF in order to calculate the potential plasmid DNA that should be r
20. ecovered after precipitation Pellet was smeared over the tube wall Dissolve DNA with an appropriate volume of Buffer TE EF by rolling the tube for at least 30 min Nucleic acid did not precipitate Check volumes of precipitating solvent making sure to use at least 0 7 volumes of isopropanol and centrifuge for longer periods of time Nucleic acid pellet does not resuspend in buffer Pellet was over dried Try dissolving at higher temperatures for a longer period of time e g 2 h at 37 C or overnight at RT best under constant spinning 3D shaker Residual salt or organic solvent in the pellet Wash the pellet with additional endotoxin free 70 ethanol or increase the resuspension buffer volume Nucleic acid pellet is Opaque or white instead of clear and glassy Salt has co precipitated with the pellet Use room temperature isopropanol and check isopropanol purity Do not precipitate by allowing the eluate to drip directly from the column into a tube containing isopropanol Add isopropanol only after eluate has been collected Centrifuge at 12 C Try resuspending the pellet in Buffer N2 EF and repeat the precipitation step MACHEREY NAGEL 03 2014 Rev 13 37 Endotoxin free plasmid DNA purification DNA is contaminated with cellular debris or genomic DNA due to inefficient lysis Reduce the culture volume or increase the amount of Buffer S1 EF S2 EF and S3 EF used during the l
21. ed by user Reagents e Isopropanol room temperatured 96 100 ethanol room temperatured Ice Equipment Standard microbiological equipment for growing and harvesting bacteria e g inoculating loop culture tubes and flasks 37 C shaking incubator and centrifuge with rotor and tubes or bottles for harvesting cells Refrigerated centrifuge capable of reaching gt 4 500 x g with rotor for the appropriate centrifuge tubes or bottles Centrifugation tubes or vessels with suitable capacity for the volumes specified in the respective protocol NucleoBond Rack Large see ordering information or equivalent holder Peristaltic pump for NucleoBond PC Prep 100 only MACHEREY NAGEL 03 2014 Rev 13 7 Endotoxin free plasmid DNA purification N Kit specifications The kits are suitable for purifying endotoxin free plasmids with lt 0 1 EU yg NucleoBond Columns are polypropylene columns containing NucleoBond AX Silica Resin packed between two inert filter elements The columns are available in several sizes to accommodate a wide range of purification needs see Table 1 Table 1 Kit specifications at a glance PC 500 EF PC2000EF PC 10000 EF PC Prep 100 Recomm medium LB Culture volume 30 150 mL 150 500 mL 500 mL 2 L 5 20 L Max pellet wet weight 0 75g 2 59 10g 90g Binding capacity 500 ug 2mg 10 mg 100 mg Endotoxin level lt 0 1 EU ug Applications Transfection studies gene therapy Time prep 100 min 150 min
22. ess genomic DNA per plasmid but they obviously work only with plasmids that do not carry the chloramphenicol resistance gene Furthermore the method is only effective with low copy number plasmids under stringent control e g PBR322 All modern high copy number plasmids e g pUC are already under relaxed control due to mutations in the plasmid copy number control genes and show no significant additional increase in their copy number Maniatis T Fritsch EF Sambrook J Molecular cloning A laboratory manual Cold Spring Harbor Cold Spring New York 1982 Frenkel L Bremer H Increased amplification of plasmids pBR322 and pBR327 by low concentrations of chloramphenicol DNA 5 539 544 1986 MACHEREY NAGEL 03 2014 Rev 13 23 NucleoBond PC EF 9 9 1 NucleoBond PC EF plasmid DNA purification Endotoxin free plasmid DNA purification Maxi Mega Giga AX 500 AX 2000 AX 10000 Cultivation and harvest of bacterial cells Set up an overnight bacterial culture by diluting an appropriate volume of starter culture into the respective volume of LB medium with selecting antibiotics Shake the culture overnight 12 16 h Centrifuge the culture at 4 500 6 000 x g for 15 min at 4 C Carefully discard the supernatant Cell lysis Carefully resuspend the pellet of bacterial cells in Buffer S1 EF RNase A Please see section 6 3 regarding difficult to lyse strains 12 mL 40 mL 120 mL
23. estions 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 lysate 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 lane 1 and 2 It might also occur in the wash fraction but must be absent in the eluate Genomic DNA should not be visible at all but would show up in the gel slot or right below indicating for example too harsh lysis conditions MACHEREY NAGEL 03 2014 Rev 13 33 Endotoxin free plasmid DNA purification u til 2a ores 5 M 1 2 w 3 dami 4 5 Marker 2 Hindlll I cleared lysate ccc linear and oc structure of the plasmid degraded RNA degraded RNA residual RNA Il lysate flow through no plasmid DNA but Ill wash flow through no plasmid DNA or IV eluate pure plasmid DNA EcoRI restriction linearized form of plasmid Figure 3 Exemplary analytical check of NucleoBond PC 500 purification samples Plasmid pUC18 bacterial strain E coli DH5a 20 uL of each precipitated sample has been analyzed on a 1 TAE agarose gel Eq
24. holen rztliche Hilfe hinzuziehen If skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltender Augenreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER 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 aufbewahren For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com A The symbol shown on labels refers to the precaution phrases of this section Das auf Etiketten dargestellte Symbol weist auf die P S tzen dieses Kapitels hin MACHEREY NAGEL 03 2014 Rev 13 21 Endotoxin free plasmid DNA purification 8 Growing of bacterial cultures 8 1 General considerations Yield and quality of plasmid DNA depend on for example the type of growing media and anti
25. ibration buffer plasmid DNA is bound to the anion exchange resin and finally eluted after efficient washing of the column Endotoxins are removed by Buffer N3 EF After precipitation of the eluted DNA it can easily be dissolved in Buffer TE EF or H O EF for further use 5 2 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 12 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification 5 3 Filtration of the lysate After alkaline lysis the solution has to be clarified from for example the cell debris through the supplied NucleoBond Folded Filters or NucleoBond Bottle Top Filters in order to prevent clogging of the column For the NucleoBond AX 2000 Mega and AX 10000 Giga Column use the supplied vacuum operated NucleoBond Bottle Top Filters for filtration of the lysate The bottle top filters Figure 1 make the separation of the bacterial lysate and SDS precipitate easy quick and convenient Adjust the
26. ins 11 4 2 Quantification of endotoxins 11 4 3 Removal of endotoxins 11 5 NuceoBond PC EF purification system 12 5 1 The basic principle 12 5 2 Convenient stopping points 12 5 3 Filtration of the lysate 13 5 4 Analytical check refers only to PC Prep 100 14 5 5 Elution procedure 17 5 6 Disposal of column resin PC Prep 100 only 17 6 Storage conditions and preparation of working solutions 18 7 Safety instructions 20 8 Growing of bacterial cultures 22 8 1 General considerations 22 8 2 Selection of culture media 22 8 3 Difficult to lyse strains 22 8 4 Chloramphenicol amplification of low copy plasmids 23 9 NucleoBond PC EF plasmid DNA purification 24 9 1 Endotoxin free plasmid DNA purification Maxi Mega Giga 24 9 2 Plasmid DNA purification preparative scale 28 MACHEREY NAGEL 03 2014 Rev 13 3 Endotoxin free plasmid DNA purification 10 Appendix 10 1 Determination of DNA yield and quality 10 2 Troubleshooting 10 3 Ordering information 10 4 References 10 5 Product use restriction warranty 32 32 32 39 40 40 4 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification 1 Components 1 1 Kit contents REF Resuspension Buffer S1 EF Lysis Buffer S2 EF Neutralization Buffer S3 EF Equilibration Buffer N2 EF Wash Buffer N3 EF Wash Buffer N4 EF Elution Buffer N5 EF Redissolving Buffer TE EF 70 EtOH Concentrate H O EF RNase A lyophilized NucleoBond AX 500 2000 10000 Col
27. ion procedure Elution is carried out into a new tube with the volume of elution buffer indicated in the corresponding protocol The plasmid DNA is precipitated by the addition of room temperature 18 25 C isopropanol Do not let the plasmid DNA solution drop into a vial with isopropanol because this leads to spontaneous co precipitation of salt Only use room temperature 18 25 C isopropanol to prevent spontaneous co precipitation of salt 5 6 Disposal of column resin PC Prep 100 only Rinse the NucleoBond AX Prep 100 Column with 350 mL of 0 2 M HCI to deactivate residual plasmid DNA and other potential biohazards Column resin or the entire column can then be disposed of according to local regulations MACHEREY NAGEL 03 2014 Rev 13 17 Endotoxin free plasmid DNA purification 6 Storage conditions and preparation of working solutions All kit components can be stored at room temperature 18 25 C and are stable for at least one years Storage of Buffer S2 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 NucleoBond PC EF prepare the following Dissolve the lyophilized RNase A by adding 1 mL of Resuspension Buffer S1 EF Pipette up and down until the RNase Ais completely dissolved Transfer the RNase
28. l tech bio mn net com MACHEREY NAGEL 03 2014 Rev 13 41
29. late is formed Incubate the suspension on ice for 5 min 4 mL Equilibration of the column Equilibrate a NucleoBond AX 100 Midi Column with Buffer N2 EF Allow the column to empty by gravity flow Discard flow through 2 5 mL Clarification of the lysate Clear the bacterial lysate by following EITHER option 1 or option 2 as described below This step is extremely important excess precipitate left in suspension may clog the NucleoBond Column in later steps Option 1 Centrifuge the suspension Centrifuge at gt 12 000xg for the minimum time indicated below at 4 C If the suspension contains residual precipitate after the first centrifugation repeat this step 25 min Option 2 Filter the suspension Place a NucleoBond Folded Filter not provided in a small funnel for support and prewet the filter with a few drops of Buffer N2 EF or sterile deionized H O Load the lysate onto the wet filter and collect the flow through Binding Load the cleared lysate from step 4 onto the NucleoBond Column Allow the column to empty by gravity flow Optional You may want to save all or part of the flow through for analysis MACHEREY NAGEL 03 2014 Rev 13 15 Endotoxin free plasmid DNA purification Midi AX 100 Washing Wash the column with Buffer N3 EF Discard flow through 10 mL Wash the column with Buffer N4 EF Discard flow through 5 mL Elution Elute the plasmid DNA with Buffer N5 E
30. mited 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 40 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability
31. night 12 16 h Centrifuge the culture at 4 500 6 000 x g for 15 min at 4 C Carefully discard the supernatant 2 Cell lysis Carefully resuspend the pellet of bacterial cells up to 90 g in Buffer S1 EF RNase A in a 4 5 L wide mouth bottle with screw closure by shaking the suspension for 5 10 minutes Alternatively stirring at a low speed about 30 rpm for one hour at RT can be performed Check that the bacterial pellet is resuspended completely and no aggregates are left 1000 mL Add Buffer S2 EF equilibrated to RT gt 20 C to avoid SDS precipitation to the suspension Immediately mix the suspension by gently inverting the flask 6 10 times resulting in a clear and very viscous solution Do not stir the resulting lysate since this may release contaminating chromosomal DNA from debris into the suspension Incubate the mixture at room temperature 18 25 C for up to 3 min 1000 mL 28 MACHEREY NAGEL 03 2014 Rev 13 NucleoBond PC Prep 100 Preparative scale AX Prep 100 Add precooled Buffer S3 EF 4 C to the suspension Immediately mix the lysate gently by inverting the flask 6 8 times until a homogeneous suspension containing an off white flocculate is formed Incubate the suspension on ice for at least 25 min 1000 mL Equilibration of the column Fix the column upright at a lab frame Equilibrate the NucleoBond AX Prep 100 Column in an upward direction with Buffer N2 EF at a flow
32. ond PC Prep 100 kits contain hazardous contents 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 up to 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze S2 EF 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 N2 EF N3 Buffer salts ethanol Warning 226 210 233 EF N4 EF 5 20 403 235 N5 EF Puffersalze Ethanol 5 20 Achtung RNase A RNase A lyophilized Danger 317 334 261 280 RNase A lyophilisiert lt gt Gefahr 302 352 304 340 333 313 342 311 363 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 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 Pre
33. or filtration e g NucleoBond Bottle Top Filters see ordering information th MACHEREY NAGEL 03 2014 Rev 13 29 NucleoBond PC Prep 100 Preparative scale AX Prep 100 5 Binding Load the cleared Iysate from step 4 onto the NucleoBond AX Prep 100 Column equilibrated with Buffer N2 EF at a flow rate of up to 4 mL min Check the real flow rate of the pump Depending on the volume of the cleared Iysate the flow rate can be reduced to get a reasonable loading time It is convenient to keep the cleared Iysate stored in an icebox during the loading of the column overnight at 3 mL min flow rate Do not cool the column itself You may want to save all or part of the flow through for analysis 6 Washing Wash the column with Buffer N3 EF at a flow rate of 15 20 mL min at room temperature 900 mL Wash the column with Buffer N4 EF at a flow rate of 15 20 mL min at room temperature 1900 mL 7 Elution Elute the plasmid DNA with Buffer N5 EF at a maximal flow rate of 4 mL min at room temperature into oven baked glassware or endotoxin free plastic vials Discard the first 200 mL of the eluate as this is the dead volume of the column The following 270 400 mL of eluate will contain the purified plasmid DNA We recommend precipitating the eluate as soon as possible step 8 Nevertheless the eluate can be stored in closed vials on ice for several hours In this case the eluate should be pre warmed to room temperatur
34. r H O EF Depending on the type of centrifugationtube dissolve under constant spinning in a sufficient amount of buffer for 10 60 min 3D shaker Determine plasmid yield by UV spectrophotometry Confirm plasmid integrity by agarose gel electrophoresis MACHEREY NAGEL 03 2014 Rev 13 31 Endotoxin free plasmid DNA purification 10 Appendix 10 1 Determination of DNA yield and quality Plasmid yield is measured by UV spectroscopy by using the following relationship 1 OD at 260 nm 1 cm path length is equivalent to 50 ug plasmid DNA mL Plasmid quality is checked initially by running a 1 agarose gel This will give information on the percentage of ccc form structural integrity of isolated plasmid DNA Plasmid quality is checked by UV spectroscopy quotient A A A value of 1 80 1 90 is an indication for pure plasmid DNA 280 Endotoxins can be measured in highly sensitive photometric tests e g Limolus Amebocyte Lysate LAL Pyrochrome Lonza Cambrex BioWhittaker and are expressed in endotoxin units EU NucleoBond PC EF PC Prep 100 purified plasmid DNA typically contains less than 0 1 EU ug Depending on further use of the purified plasmid more sophisticated analytical methods may have to be applied for quantification of byproducts 10 2 Troubleshooting If you experience problems with reduced yield or purity it is recommended to check which purification step of the procedure is cau
35. sing the problem First the bacterial culture has to be checked for sufficient growth OD in the presence of an appropriate selective antibiotic see Table 4 Second aliquots of the cleared lysate the flow through the combined washing steps Buffer N3 EF and N4 EF and the eluate should be kept for further analysis by agarose gel electrophoresis Refer to Table 3 to choose a fraction volume yielding approximately 5 ug of plasmid DNA assuming 500 ug 2000 ug 10000 ug and 100 mg were loaded onto the NucleoBond AX 500 AX 2000 AX 10000 and AX Prep 100 Column respectively Precipitate the nucleic acids by adding 0 7 volumes of isopropanol centrifuge the sample wash the pellet using 70 ethanol centrifuge again air dry for 10 minutes dissolve the DNA in 100 uL TE buffer pH 8 0 and run 20 uL on a 1 agarose gel 32 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification Table 3 NucleoBond PC EF volumes required for an analytical check Sample Purification step Volume required pL PC PC PC PC Prep 100 500 EF 2000 EF 10000 EF AX 100 check cleared lysate 400 300 200 600 after protocol step 4 Column flow ll through 400 300 200 600 after protocol step 5 m Wash flow through go 400 500 500 after protocol step 6 Eluate IN after protocol step 7 200 199 109 309 The exemplary gel picture see Figure 3 will help you to address the specific qu
36. the lysate onto the lysate onto the lysate onto the column column column _ column U 6 Washing Buffer N3 EF Buffer N3 EF Buffer N3 EF _ Buffer N3 EF 2x24 mL 1x 60 mL 4 x 150 mL Ta 900 mL 2x 40 mL u Buffer N4 EF Buffer N4 EF Buffer N4 EF Y Buffer N4 EF 2x12 mL 60 mL 3 x 130 mL 1900 mL 7 Elution Buffer N5 EF Buffer N5 EF Buffer N5 EF Buffer N5 EF 15 mL 25 mL 100 mL 470 600 mL 8 Precipitation Isopropanol Isopropanol Isopropanol Isopropanol 11 mL 18 mL 70 mL 0 7 vol 4 5 15 000 x g 4 5 15 000 x g 4 5 15 000 x g 4 5 15 000 x g 60 min at 12 C 9 Washing and drying 70 ethanol 70 ethanol 70 ethanol 70 ethanol 5 mL 7mL 10 mL 25 mL 4 5 15 000 x g 4 5 15 000 x g 4 5 15 000 x g es 4 5 15 000 x g es 10 min at RT 10 min at RT 10 min at RT 20 min at RT 10 20 min 30 60 min 30 60 min 60 min 10 Reconstitution Appropriate Appropriate Appropriate Appropriate volume of TE EF volume of TE EF volume of TE EF volume of TE EF or H O EF or H O EF or H O EF iS or H O EF 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 Endotoxin free plasmid DNA purification Table of contents 1 Components 1 1 Kit contents 1 2 Reagents and equipment to be supplied by user 2 Kit specifications 8 3 About this user manual 10 4 Endotoxins 11 4 1 Localization molecular structure and function of endotox
37. tion of the lysate and flow rate of the column will be insufficient Column overloaded with nucleic acids Use a larger column or purify excess nucleic acids on a new column Refer to the recommended culture volumes listed in No riow the table at the beginning of each protocol plasmid DNA yield Plasmid did not propagate Check plasmid content in the cleared lysate by precipitation of an aliquot Use colonies from fresh plates for inoculation and add appropriate antibiotic concentration to plates and media Alkaline lysis was inefficient If culture volume or pellet weight is too high alkaline lysis be comes inefficient Refer to the recommended culture volumes listed in Table 1 section 3 2 Lysate incorrectly prepared After storage below 20 C SDS in Buffer S2 EF may precipitate causing inefficient lysis Check Buffer S2 EF for precipitates before use and prewarm the bottle to 30 40 C if necessary in order to redissolve SDS Flow rates too high NucleoBond PC Prep 100 Do not exceed recommended flow rates for loading and eluting the plasmid DNA MACHEREY NAGEL 03 2014 Rev 13 35 Endotoxin free plasmid DNA purification Sample is too viscous Do NOT attempt to purify lysate prepared from a culture volume larger than recommended for any given column size Increasing culture volumes not only block the column but can also reduce yields due to inefficient lysis Precipitates occur during storage Ch
38. tion 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 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 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 li
39. ture 18 25 C no less than the indicated time Drying for longer periods of time will not harm the quality of plasmid DNA but overdrying may render the DNA less soluble 5 10 min 30 60 min EZ min 10 Reconstitution Dissolve pellet in an appropriate volume of Buffer TE EF or H O EF Depending on the type of centrifugationtube dissolve under constant spinning in a sufficient amount of buffer for 10 60 min 3D shaker Determine plasmid yield by UV spectrophotometry Confirm plasmid integrity by agarose gel electrophoresis MACHEREY NAGEL 03 2014 Rev 13 27 NucleoBond PC Prep 100 9 2 Plasmid DNA purification preparative scale The following NucleoBond PC Prep 100 protocol is based on the application of 10 liters of LB culture corresponding to up to 90 g of bacterial cells The NucleoBond AX Prep 100 Column is for single use only Pressure within the column should not exceed 7 kg cm 100 PSI during usage Use only oven baked glassware or new disposable plasticware for handling the purified plasmid DNA to prevent contamination with endotoxins This is important for the elution precipitation and reconstitution step Preparative scale AX Prep 100 1 Cultivation and harvest of bacterial cells Set up an overnight bacterial culture by diluting an appropriate volume of starter culture into the respective volume of LB medium with selecting antibiotics Shake the culture over
40. ual amounts of plasmid DNA before lane 1 and after lane 4 purification using NucleoBond PC 500 are shown with a recovery of gt 90 Table 4 Information about antibiotics according to Maniatis Antibiotic Stock 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 g 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 g mL Tetracycline 5 mg mL in EtOH 20 C 10 50 g mL Maniatis T Fritsch EF Sambrook J Molecular cloning A laboratory manual Cold Spring Harbor Cold Spring New York 1982 34 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification Problem Possible cause and suggestions SDS or other precipitates are present in the sample Load the S1 EF S2 EF S3 EF lysate sample onto the NucleoBond Column immediately after finishing the initial lysis steps SDS and cell debris are removed by filtration with NucleoBond Folded Filters NucleoBond Bottle Top Filters but if the cleared lysate is stored on ice for a longer period new precipitate may appear If precipitate is visible it is recommended to filter the lysate again immediately before loading it onto the NucleoBond Column Sample lysate is too viscous Watch maximal volumes and pellet wet weights given in the manual Otherwise filtra
41. umns NucleoBond Folded Filters NucleoBond Bottle Top Filters Type 1 or 2 Plastic Washers User manual NucleoBond PC 500 EF 10 preps 740550 150 mL 150 mL 150 mL 100 mL 800 mL 350 mL 200 mL 30 mL 35 mL 30 mL 15 mg 10 10 NucleoBond PC 2000 EF 5 preps 740549 250 mL 250 mL 250 mL 150 mL 800 mL 350 mL 200 mL 30 mL 35 mL 30 mL 25 mg For preparation of working solutions and storage conditions see section 4 NucleoBond PC 10000 EF 5 preps 740548 750 mL 750 mL 750 mL 600 mL 4 x 800 mL 2 x 1000 mL 600 mL 30 mL 35 mL 30 mL 75mg MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification 1 1 Kit contents continued NucleoBond PC Prep 100 1 prep REF 740594 Resuspension Buffer S1 EF 1000 mL Lysis Buffer S2 EF 1000 mL Neutralization Buffer S3 EF 1000 mL Equilibration Buffer N2 EF 1000 mL Wash Buffer N3 EF 1000 mL Wash Buffer N4 EF 2 x 1000 mL Elution Buffer N5 EF 600 mL Redissolving Buffer TE EF 60 mL 70 EtOH Concentrate 2x35mL H O EF 60 mL RNase A lyophilized 100 mg NucleoBond AX Prep 100 Column 1 NucleoBond AX 100 Columns 3 Sieving Fabric 3 NucleoBond Folded Filters 2x5 Type 1 and 2 User manual 1 For preparation of working solutions and storage conditions see section 4 6 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification 1 2 Reagents and equipment to be suppli
42. umns 740547 5 2 NucleoBond Bottle Top Filters Type 2 for NucleoBond AX 10000 Columns 740553 5 5 NucleoBond Rack Large for NucleoBond AX 100 500 2000 and 740563 1 10000 Columns RNase A lyophilized 740505 50 50 mg 740505 100 mg Visit www mn net com for more detailed product information MACHEREY NAGEL 03 2014 Rev 13 39 Endotoxin free plasmid DNA purification 10 4 References Birnboim H C and Doly J 1979 Nucl Acids Res 7 1513 1523 10 5 Product use restriction warranty NucleoBond EF kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purifica
43. ysis steps Purified a ER d DNA is degraded does not Make sure that all equipment pipettors centrifuge tubes etc perform well are clean and nuclease free Make sure that the alkaline lysis insubseanenf step i e the incubation of sample after addition of Buffer S2 q EF does not proceed for longer than 5 min reactions Washing steps inefficient NucleoBond PC Prep 100 Wash the column extensively with Buffer N3 EF and N4 EF respectively until UV absorbance has reached the initial value of equilibration Culture volumes used are too large NucleoBond Reduce the culture volume or increase the amount of Buffer ue e001 S1 EF S2 EF and S3 EF used during the lysis steps Folded Filters clog during SAN filtration Incubation time too short Make sure that S1 EF S2 EF S3 EF lysate was incubated according to the protocol 38 MACHEREY NAGEL 03 2014 Rev 13 Endotoxin free plasmid DNA purification 10 3 Ordering information Product REF Pack of NucleoBond PC 500 EF 740550 10 preps NucleoBond AX 500 740531 10 columns NucleoBond AX 500 740531 50 50 columns NucleoBond PC 2000 EF 740549 5 preps NucleoBond AX 2000 740525 10 columns NucleoBond PC 10000 EF 740548 5 preps NucleoBond AX 10000 740534 5 columns NucleoBond PC Prep 100 740594 1 prep NucleoBond Folded Filters XL for NucleoBond AX 500 Columns 740577 50 NucleoBond Bottle Top Filters Type 1 for NucleoBond AX 2000 Col
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