Plasmid DNA Purification
Contents
1. Problem Possible cause and suggestions Pellet was over dried e Try dissolving at temperatures for a longer period of time e g Nuci ieseid 2 h at 37 C or overnight at RT best under constant spinning 3D shaker pellet will not resuspend in buffer There is residual salt or organic solvent in the pellet e Wash the pellet with additional low viscosity organic solvent 70 ethanol or increase the resuspension buffer volume Salt has co precipitated with the pellet Check isopropanol purity and perform precipitation at room temperature 20 25 C but centrifuge at 4 C Do not precipitate eren acid by allowing the eluate to drip directly from the column into a tube AAG Ok containing isopropanol Add isopropanol only after eluate has paqu been collected white instead A and e Try resuspending the pellet in buffer N2 and reload onto the grassy NucleoBond column Be sure to wash the column several times with buffer N2 before loading the redissolved pellet onto the col umn DNA is contaminated with cellular debris or genomic DNA due to inefficient lysis e Reduce the culture volume or increase the amount of buffers Purified plas 1 S2 and S3 used during the lysis steps Mis Wene A perform well in subsequent DNA is degraded l reactions Make sure that all equipment pipettes centrifuge tubes etc are clean and nuclease free Make sure that the alkaline lysis step i e the incubation of sample after a2. 100 ml MACHEREY NAGEL 03 2005 Rev 03 23 Plasmid DNA Purification 24 AX 2000 AX 10000 Clarification of the lysate Clear the lysate by following EITHER option 1 or option 2 described below This step is extremely important excess precipitate left in suspension may clog the NucleoBond column in later steps Option 1 Filter the suspension Place a NucleoBond folded filter in a large funnel for support Prewet the filter with a few drops of buffer N2 or sterile deionized HO and load lysate Note NucleoBona PC 10000 Giga kits contain two types of folded filters Type 1 and Type 2 in order to guarantee an optimal and fast filtration Put folded filter Type 2 into folded filter Type 1 and prewet the filters with a few drops of buffer N2 or sterile deion ized H O before loading lysate For Giga Columns we recommend dividing the samples in half to clear the lysate use two NucleoBond folded filters and two funnels simultaneously Then combine the flow through before proceeding with step 5 For AX 2000 Mega and AX 10000 Giga columns alternatively the vacuum operated NucleoBond bottle top filters not included can be used for filtration of the lysate For correct use of the NucleoBond bottle top filters see section 3 5 Alternative Option 2 Centrifuge the suspension Centrifuge at gt 12 000 x g for the minimum time indicated below at 4 C If the suspension contains residual precipitate after th
3. AX 2000 1 Cultivate and harvest bacterial cells Harvest bacteria from an LB culture by centrifugation at 4 500 6 000 x g for 15 min at 4 C 2 Cell lysis Carefully resuspend the pellet of bacterial cells in buffer S1 RNase A Please see section 6 3 regarding difficult to lyse strains Add buffer S2 to the suspension Mix gently by inverting the tube 6 8 times In cubate the mixture at room temperature 20 25 C for 2 3 min max 5 min Do not vortex as this will release contaminating chromosomal DNA from the cellular debris into the suspension Add pre cooled buffer S3 4 C to the suspension Immediately mix the lysate gently by inverting the flask 6 8 times until a homogeneous suspension contain ing an off white flocculate is formed Incubate the suspension on ice for 5 min 3 Equilibration of the column Equilibrate a NucleoBond AX 500 Maxi BAC 100 Maxi or AX 2000 Mega column with buffer N2 Allow the column to empty by gravity flow Discard flow through MACHEREY NAGEL 03 2005 Rev 03 29 Plasmid DNA Purification 30 AX 500 BAC 100 AX 2000 Clarification of the lysate Clear the lysate by following EITHER option 1 or option 2 described below This step is extremely important excess precipitate left in suspension may clog the NucleoBond column in later steps Note for purification of BAC DNA it is recommended to follow option 1 Option 1 Filter the suspension Place a
4. Plasmid DNA Purification User manual NucleoBond PC 20 NucleoBond PC 100 NucleoBond PC 500 NucleoBond BAC 100 NucleoBond PC 2000 NucleoBond PC 10000 March 2005 Rev 03 MACHEREY NAGEL MN Protocol at a glance Rev 03 Plasmid DNA Purification Mini Midi Maxi Mega Giga 10 step Cultivate and harvest AX 20 4 500 6 000 x g Midi AX 100 4 500 6 000 x g AX 500 4 500 6 000 x g AX 2000 4 500 6 000 x g Giga AX 10000 4 500 6 000 x g 15 min at 4 C 15 min at 4 C 15 min at 4 C 15 min at 4 C 15 min at 4 C bacterial cells Cell lysis high copy low copy Buffer S1 0 4 mil 0 8 ml 4 ml 8 ml 12 ml 24 ml 45 mil 90 ml 120 ml Buffer S2 0 4 mil 0 8 ml 4 ml 8 ml 12 ml 24 ml 45 mil 90 ml 120 ml Buffer S3 0 4 ml 0 8 ml 4 ml 8 ml 12 ml 24 ml 45 mil 90 ml 120 ml Fqullibrationof Buffer N2 Buffer N2 Buffer N2 Buffer N2 Buffer N2 1 ml 2 5 ml 6 0 ml 20 ml 100 ml ji Clarification of Centrifugation Folded filter Folded filter Folded filter Folded filter the lysate or or or or A centrifugation centrifugation centrifugation centrifugation 15 min 25 min 40 min 50 min 60 min 12 000 xg 12 000 xg 12 000 xg 12 000 xg 12 000 xg Binding Load cleared Load cleared Load cleared Load cleared Load cleared lysate onto the lysate onto the lysate onto the lysate onto the lysate onto the column column column column column 7 Buffer N3 Buffer N3 B
5. ters avoid shearing of large DNA constructs such as PACs or BACs Folded Filters should not be used with AX 20 Mini columns for plas mid preparation due to the small culture volumes which allow an easy and quick clearing of the lysate in a microcentrifuge Due to the large cul ture volume two folded fiters Type 1 and Type 2 are included in the NucleoBond PC 10000 kit in order to guarantee an optimal removal of SDS and cellular debris from plasmid sample For correct use please follow the instructions given in step 4 of the corresponding protocol Alternatively Figure 2 Correct use of the folded filters Centrifuge the solution with the given acceleration forces and times as written in step 4 of the corresponding protocols Load the cleared lysate onto the col umn For the AX 2000 Mega column and AX 10000 Giga column also the vacuum operated Nu cleoBond bottle top filters can be used for fil tration of the lysate The NucleoBond bottle top filters Figure 3 make the separation of the bacterial lysate and SDS precipitate easily quickly and conveniently When using a Nu cleoBond bottle top filter it is not necessary to centrifuge the solution first as described in step 4 option 1 and 2 of the corresponding protocol Adjust the bottle top filter to a suitable tlask e g Schott load the bacterial lysate and apply the vacuum After 3 5 min the solution will have passed through Load the resulting clear
6. 5 30 ml AX 100 Midi 20 100 ug 30 150 ml 0 75 g AX 500 Maxi 100 500 ug 150 500 ml 2 5 g AX 2000 Mega 500 ug 2 mg 500 2 000 ml 10g AX 10000 Giga 2 mg 10 mg Low copy 3 10 ml AX 20 Mini 3 20 ug 1 100 ml AX 100 Midi 20 100 ug 100 500 ml 1 5 2 2 g AX 500 Maxi 100 500 ug 100 500 ml 1 5 2 2 g BAC 100 Maxi 100 ug 500 2 000 ml 5 7 5g AX 2000 Mega 500 ug 2 mg For AX 20 and AX 100 it is not necessary to measure the wet weight but depending on the media used ODgo should be determined For a low copy protocol using AX 10000 Giga columns please call our Technical Service Center 18 MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification 6 2 Selection of culture media The cultivation of cells is recommended at 37 C in LB Luria Bertani medium at con stant shaking 200 250 rpm Alternatively rich media like 2xYT 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 s 12 h This may lead to a higher percentage of dead or starving cells when starting the preparation The re sulting plasmid DNA from overgrown cultures may be partially degraded or contami nated with chromosomal DNA For Mini and Midi preps cultivation in flasks is recommended At least for Mega and Giga preps the use of an appropriate fermentation system is recommended in order to optimize cultivation conditions 6 3 Difficult t
7. DNA ml Plasmid quality is checked initially by running a 1 agarose gel This will give information on percentage of ccc form structural integrity of isolated plasmid DNA e Plasmid quality is checked by UV spectroscopy quotient 260 nm 280 nm A value of 1 80 1 90 is an indication for pure plasmid DNA Depending on further use of the purified plasmid more sophisticated analytical methods may have to be applied for quantification of byproducts 8 2 Troubleshooting If you experience problems with reduced yield or purity it is recommended to check at which purification step of the procedure the problem occured Firstly the bacterial culture has to be checked for sufficient growth OD 9 in the presence of an appro priate selective antibiotic see Table 4 Secondly aliquots of the cleared lysate the flow through the combined washing steps buffer N3 and the eluate should be kept for further analysis by agarose gel electrophoresis Refer to Table 3 to choose a fraction volume yielding approximately 5 yg of plasmid DNA The volumes outlined in Table 3 refer to maximum yield binding capacity of each column size used for the preparation please also see Tables 1 and 2 Pre cipitate the nucleic acids by adding 0 7 volumes of isopropanol centrifuge the sam ple wash the pellet using 70 ethanol centrifuge again air dry for 10 minutes dis solve the DNA in 100 ul TE buffer pH 8 0 and run 20 ul on a 1 agarose gel The gel p
8. NucleoBond folded filter in a large funnel for support Prewet the filter with a few drops of buffer N2 or sterile deionized HO Shortly spin down the lysate at low g force in order to let the cellullar debris settle before loading on the NucleoBond folded filter When the centrifuge has stopped carefully decant the partially cleared lysate onto the wet filter and collect the flow through For the AX 2000 Mega column alternatively the vacuum operated NucleoBond bottle top filters not included can be used for filtration of the lysate Alternative Option 2 Centrifuge the suspension Centrifuge at gt 12 000 x g for the minimum time indicated below at 4 C If the suspension contains residual precipitate after the first centrifugation either repeat this step or proceed with option 1 Binding Load the cleared lysate from step 4 onto the NucleoBond column Allow the col umn to empty by gravity flow You may want to save all or part of the flow through for analysis Washing Wash the column with buffer N3 Repeat as indicated Discard flow through MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification 10 AX 500 BAC 100 AX 2000 Elution Elute the plasmid DNA with buffer N5 Preheating buffer N5 to 50 C prior to elution may improve yields for high molecular weight constructs such as BACs We recommend precipitating the eluate as soon as possible step 8 Nevertheless the eluate can be stored in cl
9. but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connec tion with the sale or the failure of MACHEREY NAGEL products to perform in accor dance 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 cata logues and product literature are MACHEREY NAGEL s sole representations con cerning 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 author ized they should not be relied upon by the customer and are not a part of the con tract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Serv ice 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 MA CHEREY NAGEL does not warrant the correctnes
10. lysate from step 4 onto the NucleoBond column Allow the col umn to empty by gravity flow Optional You may want to save all or part of the flow through for analysis Washing Wash the column with buffer N3 Repeat as indicated Discard flow through wn EXI MACHEREY NAGEL 03 2005 Rev 03 21 Plasmid DNA Purification 10 22 7 KI AX 20 AX 100 AX 500 Elution Elute the plasmid DNA with buffer N5 We recommend precipitating the eluate as soon as possible step 8 Neverthe less the eluate can be stored in closed vials on ice for some hours In this case the eluate should be prewarmed to room temperature before the plasmid DNA is precipitated Optional Determine plasmid yield by UV spectrophotometry in order to adjust the de sired concentration of DNA step 10 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Mix carefully and centrifuge at 15 000 x g for 30 min at 4 C Carefully discard the supernatant 075 mi 3 5 mi 11 0 mi Wash and dry DNA pellet Add room temperature 70 ethanol to the pellet Vortex briefly and centrifuge at 15 000 x g for 10 min at room temperature 20 25 C Carefully remove ethanol from the tube with a pipette tip Allow the pellet to dry at room temperature 20 25 C no less than the indicated time Drying for longer periods will not harm the quality of the plasmid DNA Reconstitute DNA Redissolve the DNA pellet in an
11. on the pH value of the eluent For this reason pH values must be carefully controlled if the buffers have been prepared by the customer A deviation of more than 0 1 pH unit from the given values may affect yields If you are consistently expe riencing reduced product yields check the pH of all buffers before continuing Buffers should be adjusted with H PO or KOH 12 MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification 3 4 High low copy plasmid purification NucleoBond PC kits are recommended for the isolation of high copy plasmids gt 20 copies cell however low copy plasmids lt 20 copies cell can be isolated as well If you are purifying low copy plasmids you will need to supplement the NucleoBond PC kits with additional buffers We recommend the NucleoBond Buffer Set I Cat No 740601 for routine purification of low copy plasmids The NucleoBond Buffer Set I can be used in connection with NucleoBond PC kits for the isolation of low copy plasmids In this combination it is sufficient for e NucleoBond PC 500 kit Cat No 740574 10 preparations low copy plas mid purification e NucleoBond PC 100 kit Cat No 740573 20 preparations low copy plas mid purification e NucleoBond PC 20 kit Cat No 740571 100 100 preparations low copy plasmid purification In connection with NucleoBond AX columns the NucleoBond Buffer Set I can be used for the isolation of high copy plasmids In this combi
12. overloaded with nucleic acids plasmid DNA yield Use a larger column or purify excess nucleic acids on a new column Refer to the recommended culture volumes listed in the table at the beginning of each protocol Plasmid did not propagate Check plasmid content in the cleared lysate by precipitation of an aliquot Use colonies from fresh plates for inoculation and add appropriate antibiotic concentration to plates and media Alkaline lysis was inefficient If culture volume or pellet weight is too high alkaline lysis be comes inefficient Refer to the recommended culture volumes listed in Table 2 section 6 1 Lysate incorrectly prepared After storage below 20 C SDS in buffer S2 may precipitate This will lead to a suboptimal SDS concentration in buffer S2 causing inefficient lysis Check buffer S2 for precipitates before use and prewarm the bottle if necessary in order to redissolve SDS 30 40 C will be sufficient MACHEREY NAGEL 03 2005 Rev 03 35 Plasmid DNA Purification Problem Possible cause and suggestions 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 blocks the column but also significantly reduces yields Precipitates occur during storage Column is blocked Check cleared lysate for precipitates especially if the lysate was stored for a longer time before loadin
13. to accommodate a wide range of purification needs see Ta ble 1 NucleoBond column Binding capacity AX 20 20 ug AX 100 100 pg AX 500 500 ug BAC 100 500 ug AX 2000 2 mg AX 10000 10 mg MACHEREY NAGEL 03 2005 Rev 03 11 Plasmid DNA Purification All NucleoBond columns are resistant to organic solvents such as alcohol chloroform and phenol and are free of DNase and RNase e 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 3 3 Buffer compositions Buffer S1 e 50 mM Tris HCl 10 mM EDTA 100 ug ml RNase A pH 8 0 Buffer S2 200 mM NaOH 1 SDS Buffer S3 e 2 8 M KAc pH 5 1 Buffer N2 100 mM Tris 15 ethanol 900 mM KCI 0 15 Triton X 100 adjusted to pH 6 3 with HPO Buffer N3 100 mM Tris 15 ethanol 1 15 M KCI adjusted to pH 6 3 with H PO Buffer N5 e 100 mM Tris 15 ethanol 1 M KCl adjusted to pH 8 5 with H PO Note Keep all buffers tightly capped The concentration of KCI required for eluting the desired nucleic acid is highly de pendent
14. 00 ml RNase A lyophilized 12 mg 2x 25mg NucleoBond AX 100 columns 20 100 NucleoBond folded filters 20 100 Plastic washer 10 10 Protocol 1 1 For preparation of working solutions and storage conditions see section 4 MACHEREY NAGEL 03 2005 Rev 03 5 Plasmid DNA Purification 1 Kit contents continued Cat No Buffer S1 Buffer S2 Buffer S3 Buffer N2 Buffer N3 Buffer N5 RNase A lyophilized NucleoBond AX 500 columns NucleoBond folded filters Plastic washer Protocol 10 preps 740574 150 ml 150 ml 150 ml 70 ml 2 x 250 ml 200 ml 15 mg 10 10 25 preps 740574 25 2 x 200 ml 400 ml 400 ml 200 ml 2 x 500 ml 500 ml 2x 25mg 25 25 50 preps 740574 50 2 x 400 ml 2 x 400 ml 2 x 400 ml 2 x 200 ml 2 x 1000 ml 2 x 500 ml 2x 40mg 50 50 10 For preparation of working solutions and storage conditions see section 4 6 MACHEREY NAGEL 03 2005 Rev 03 100 preps 740574 100 3 x 500 ml 3 x 500 ml 3 x 500 ml 4x 200 ml 3 x 1000 ml 500 ml 3 x 500 ml 200 ml 3 x 50 mg 100 100 10 1 Plasmid DNA Purification 1 Kit contents continued 5 preps 5 preps Cat No 740576 740593 Buffer S1 250 ml 750 ml Buffer S2 250 ml 750 ml Buffer S3 250 ml 750 ml 500 ml Buffer N2 140 ml 120 ml 1000 ml Buffer N3 2 x 250 ml 2 x 200 ml 500 ml Buffer N5 200 ml 120 ml RNase A lyophilized 20 109 soma Nucl
15. AX 100 columns 38 MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification Product Cat No Pack of NucleoBond folded filters XL for AX 500 2000 BAC 100 columns 740577 50 NucleoBond bottle top filters for AX 2000 10000 columns Or request NucleoBond buffer set 740601 1 set NucleoBond buffer S1 740516 1 500 ml NucleoBond buffer S2 740517 1 500 ml NucleoBond buffer S3 740518 1 500 ml NucleoBond buffer N2 740527 1 500 ml NucleoBond buffer N3 740528 1 1000 ml NucleoBond buffer N5 740529 1 500 ml NucleoBond rack small for AX 20 columns 40902 NucleoBond rack large for AX 100 AX 500 AX 2000 740563 1 AX 10000 columns RNase A 740505 100 mg RNase A 740505 50 50 mg 8 4 References Birnboim H C and Doly J 1979 Nucl Acids Res 7 1513 1523 8 5 Product use restriction warranty NucleoBond PC BAC kit components were developed designed and sold for re search purposes only They are suitable for in vitro uses only No claim or repre sentation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoBond PC BAC kit for a specific application range as the performance characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifica tions and
16. acterial cultures 6 1 General considerations 6 2 Selection of culture media 6 3 Difficult to lyse strains NucleoBond plasmid purification 7 1 General procedure 7 2 High copy plasmid purification Mini Midi Maxi 7 3 High copy plasmid purification Mega Giga 7 4 Low copy plasmid purification Mini Midi 7 5 Low copy plasmid purification Maxi BAC Mega Appendix 8 1 Determination of DNA yield and quality 8 2 Troubleshooting 8 3 Ordering information 8 4 References 8 5 Product use restriction warranty MACHEREY NAGEL 03 2005 Rev 03 10 11 11 11 12 13 14 15 16 17 18 18 19 19 20 20 20 23 26 29 32 32 32 38 39 39 Plasmid DNA Purification 1 Kit contents 20 preps 100 preps Cat No 740571 740571 100 Buffer S1 20 ml 2x 35 ml Buffer S2 20 ml 2x 35 ml Buffer S3 20 ml 2x 35 ml Buffer N2 25 ml 125 ml Buffer N3 2x 35 ml 3 x 125 ml Buffer N5 35 ml 125 ml RNase A lyophilized 2 mg 2x 4 mg NucleoBond AX 20 columns 20 100 NucleoBond folded filters Plastic washer 10 10 Protocol 1 1 For preparation of working solutions and storage conditions see section 4 4 MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification 1 Kit contents continued 20 preps 100 preps Cat No 740573 740573 100 Buffer S1 120 ml 2 x 250 ml Buffer S2 120 ml 2 x 250 ml Buffer S3 120 ml 2 x 250 ml Buffer N2 70 ml 2 x 150 ml Buffer N3 240 ml 3 x 400 ml Buffer N5 120 ml 3 x 2
17. appropriate volume of buffer TE or sterile deion ized HO Depending on the type of centrifugation tube redissolve under con stant 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 2005 Rev 03 Plasmid DNA Purification 7 3 High copy plasmid purification Mega Giga Giga AX 2000 AX 10000 1 Cultivate and harvest bacterial cells Harvest bacteria from an LB culture by centrifugation at 4 500 6 000 x g for 15 min at 4 C 2 Cell lysis Carefully resuspend the pellet of bacterial cells in buffer S1 RNase A Please see section 6 3 regarding difficult to lyse strains 120 mi Add buffer S2 to the suspension Mix gently by inverting the tube 6 8 times In cubate the mixture at room temperature 20 25 C for 2 3 min max 5 min Do not vortex as this will release contaminating chromosomal DNA from the cellular debris into the suspension 120 mi Add pre cooled buffer S3 4 C to the suspension Immediately mix the lysate gently by inverting the flask 6 8 times until a homogeneous suspension contain ing an off white flocculate is formed Incubate the suspension on ice for 5 min 120 mi 3 Equilibration of the column Equilibrate a NucleoBond AX 2000 Mega AX 10000 Giga column with buffer N2 Allow the column to empty by gravity flow Discard flow through
18. below 25 g or ml concerning 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 42 and TRGS 200 7 1 MACHEREY NAGEL 03 2005 Rev 03 17 Plasmid DNA Purification 6 Growing of bacterial cultures 6 1 General considerations Yield and quality of plasmid DNA depends on e g the type of growing media and an tibiotics the bacterial host plasmid type size or copy number Therefore these factors should be taken into consideration For cultivation of bacterial cells we rec ommend LB medium The suggested bacterial culture volumes for each column size as well as expected plasmid yields are listed in Table 2 Overnight cultures in flasks usually reach under vigorous shaking an ODs of 3 6 while fermentation cultures reach 10 and more Therefore please refer not only to the culture volume but also check ODs and pellet wet weight too in particular if richer culture media like 2xYT or TB are used If too much bacterial material is used lysis and precipitation steps are inefficient and finally NucleoBond columns are overloaded causing decreased yield and plasmid quality As a general rule 1 liter E coli culture grown in LB medium yields a pellet of about 3 20 g wet weight The expected yield for a high copy number plasmid is 1 3 mg per gram wet weight Table 2 Recommended culture volume Copy plasmids LB culture Wet weight Recommended Average yield volume of pellet column size High copy 1 5 ml AX 20 Mini 3 20 ug
19. cleoBond Plasmid purification kit may refer to the Protocol at a glance instead of this user manual The Protocol at a glance is designed to be used only as a sup plemental tool for quick referencing while performing the purification procedure First time users are strongly advised to read this user manual The protocols in this manual are organized as follows The volumes of the respective buffers used for a particular column size are high lighted Each procedural step is arranged like the following example taken from sec tion 7 2 High copy plasmid purification 7 AX 20 AX 100 AX 500 1 Carefully resuspend the pellet of bacterial cells in buffer S1 RNase A Please see section 6 3 regarding difficult to lyse strains vid a For example if you are performing a Mini Prep to purify plasmid DNA using an AX 20 column you are requested to refer to the white boxes In these boxes there are noted the volumes of buffers to be used The name of the buffer the indicated volume is referring to is highlighted in bold type within the instruction Referring to the a m example the pellet of the bacterial cells has to be resuspended in 0 4 ml of buffer S1 when performing a Mini prep using NucleoBond AX 20 col umns 10 MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification 3 Product description 3 1 The basic principle NucleoBond PC BAC Kits employ a modified alkaline SDS lysis procedure to pre pare the ba
20. cterial 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 chro mosomal DNA and other cellular compounds The potassium acetate buffer also neutralizes the lysate Plasmid DNA which remains in solution can revert to its na tive supercoiled structure After equilibrating the appropriate NucleoBond column with equilibration buffer plasmid DNA is bound to the anion exchange resin and fi nally eluted after efficient washing of the column After precipitation of the eluted DNA it can easily be dissolved in TE buffer for further use 3 2 Kit specifications NucleoBond Plasmid Purification Kits contain NucleoBond columns ap propriate buffers and RNase A Kits are available for each column size Mini PC 20 Midi PC 100 Maxi PC 500 BAC 100 Mega PC 2000 and Giga PC 10 000 e The protocols are suitable for purifying most plasmids ranging from 3 gt 10 kb cosmids from 10 50 kb and very large constructs P1 constructs BACs PACs up to 300 kb NucleoBond columns are polypropylene columns containing NucleoBond AX silica resin packed between two inert filter elements NucleoBond col umns are used to purify plasmids cosmids P1 constructs BACs PACs and phage DNA not described in this user manual The columns are available in several sizes
21. ddition of buffer S2 does not proceed for longer than 5 min NucleoBond folded filters clog during filtration Culture volumes used are too large Reduce the culture volume or increase the amount of buffers S1 S2 and S3 used during the lysis steps Incubation time too short Make sure that S1 S2 S3 lysate was incubated according to the protocol MACHEREY NAGEL 03 2005 Rev 03 37 Plasmid DNA Purification 8 3 Ordering information Product Cat No Pack of NucleoBond PC 20 740571 20 preps NucleoBond PC 20 740571 100 100 preps NucleoBond AX 20 740511 20 columns NucleoBond PC 100 740573 20 preps NucleoBond PC 100 740573 100 100 preps NucleoBond AX 100 740521 20 columns NucleoBond AX 100 big pack 740521 100 100 columns NucleoBond PC 500 740574 10 preps NucleoBond PC 500 740574 25 25 preps NucleoBond PC 500 740574 50 50 preps NucleoBond PC 500 740574 100 100 preps NucleoBond AX 500 740531 10 columns NucleoBond AX 500 big pack 740531 50 50 columns NucleoBond PC 2000 740576 5 preps NucleoBond AX 2000 740525 10 columns NucleoBond PC 10000 740593 5 preps NucleoBond AX 10000 740534 5 columns NucleoBond Finalizer includes 20 NucleoBond Finalizer 740519 20 20 filters 2 syringes of 30 ml 2 syringes of 1 ml NucleoBond Finalizer includes 20 NucleoBond Finalizer 740520 20 20 sets 20 syringes of 30 ml 20 syringes of 1 ml NucleoBond folded filters 740561 50 for
22. e first centrifugation either repeat this step or proceed with option 1 Binding Load the cleared lysate from step 4 onto the NucleoBond column Allow the col umn to empty by gravity flow You may want to save all or part of the flow through for analysis Washing Wash the column with buffer N3 Repeat as indicated Discard flow through MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification Giga AX 2000 AX 10000 10 Elution Elute the plasmid DNA with buffer N5 We recommend precipitating the eluate as soon as possible step 8 Neverthe less the eluate can be stored in closed vials on ice for some hours In this case the eluate should be prewarmed to room temperature before the plasmid DNA is precipitated 100 ml Optional Determine plasmid yield by UV spectrophotometry in order to adjust desired concentration of DNA step 10 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Cen trifuge at 15 000 x g for 30 min at 4 C Carefully discard the supernatant 70 mi Wash and dry DNA pellet Add room temperature 70 ethanol to the pellet Vortex briefly and centrifuge at gt 15 000 x g for 10 min at room temperature 20 25 C 7 mi 10 ml Carefully remove ethanol from the tube with a pipette tip Allow the pellet to dry at room temperature 20 25 C Drying for longer periods will not harm the quality of the plasmid DNA 30 60 min Reconstit
23. eoBond 5 j AX 2000 columns NucleoBond j 5 AX 10000 columns NucleoBond folded filters 9 10 Plastic washer 5 Protocol 1 1 For preparation of working solutions and storage conditions see section 4 MACHEREY NAGEL 03 2005 Rev 03 7 Plasmid DNA Purification 1 Kit contents continued Cat No Buffer S1 Buffer S2 Buffer S3 Buffer N2 Buffer N3 Buffer N5 RNase A lyophilized NucleoBond BAC 100 columns NucleoBond folded filters Plastic washer Protocol 10 preps 740579 2x 150 ml 2x 150 ml 2x 150 ml 70 ml 2 x 200 ml 150 ml 2x 15mg 10 10 For preparation of working solutions and storage conditions see section 4 8 MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification 2 Introduction 2 1 Properties NucleoBond AX is a silica based anion exchange resin developed by MACHEREY NAGEL for routine separation of different classes of nucleic acids Nu cleoBond AX resin covered under European Patent EP 0496822 forms the basis for the entire line of nucleic acid purification products presented in this User Manual NucleoBond AX resin consists of hydrophilic macro porous silica beads coupled to a methyl ethylamine functional group The functional group provides a high overall charge density that permits the negatively charged phosphate backbone of plasmid DNA to bind with high specificity to the resin Due to a specialized manufacturing process that is rigorousl
24. g If necessary clear the lysate again by filtration Lysate was not completely cleared Centrifuge at higher speed or for a longer period of time or use additional NucleoBond folded filters to clear the lysate Lysis treatment was too harsh Be sure not to incubate the lysate in buffer S2 for more than 5 min Overzealous mixing during lysis allowed genomic DNA to shear off Sa YA into the lysis buffer tamination of If the lysate is too viscous to mix properly or gently reduce cul plasmid DNA ture volumes RNase digestion was inefficient e RNase was not added to buffer S1 or stored too long Add new RNase to buffer S1 See ordering information section 8 3 Pellet was lost Handle the precipitate with care Decant solutions carefully Measure DNA yield in buffer N5 in order to calculate the poten tial plasmid DNA that should be recovered after precipitation i Pellet did not resuspend in buffer aria pellet e Again handle the pellet with care Especially if the DNA was formed after precipitation precipitated in a gt 15 ml tube the pellet may be smeared over the wall of the tube Dissolve DNA with an appropriate volume of TE buffer by rolling the tube for at least 30 min 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 peri ods of time 36 MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification
25. h RNase A at 4 C The solution will be stable at this temperature up to 6 months Buffer S2 should be stored at room temperature 20 25 C since the contain ing SDS may precipitate at temperatures below 20 C If precipitation occurs incubate the bottle for several minutes at about 30 40 C and mix well until the precipitate is redissolved 16 MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification 5 Safety instructions risk and safety phrases The following components of the NucleoBond PC kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section RNase A Xn May cause sensitization by inhalation R 42 43 S 7 16 22 RNase A lyophilized x and skin contact sodium gt xj Irritating to eyes and skin R 36 38 S 22 24 S2 hydroxide x 26 36 37 lt 2 Risk Phrases R 36 38 Irritating to eyes and skin R 42 43 May cause sensitization by inhalation and skin contact Safety Phrases S7 Keep container tightly closed S16 Keep away from sources of ignition No Smoking S 22 Do not breathe dust S 24 Avoid contact with the skin S 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S 36 37 Wear suitable protective clothing and gloves Label not necessary if quantity below 125 g or ml concerning 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 42 and TRGS 200 7 1 Label not necessary if quantity
26. icture see Figure 4 will help you to address the specific questions outlined in this section more quickly and efficiently 32 MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification Table 3 NucleoBond PC volumes required for analytical check Sample Purification step Volume required pl PC 100 PC 500 PC 2000 PC 10000 Cleared Weal 600 400 300 200 after protocol step 4 Flow through 7 after protocol step 5 600 400 300 00 ll Washing flow through 500 300 200 100 after protocol step 6 Eluate IV after protocol step 7 ae dk 109 me Figure 4 Analytical check of NucleoBond PC 500 purification samples Plasmid pUC18 bacterial strain E coli DH5a 20 ul of each sample has been analyzed ona 1 agarose gel Equal amounts of plasmid DNA before lane 1 and after lane 4 purification using NucleoBond PC 500 are shown with a recovery of gt 90 12345 mt Marker Hindlll Cleared lysate ccc linear and oc structure of the plasmid degraded RNA Il Flow through no plasmid DNA but degraded RNA Ill Washing flow through no plasmid DNA or residual RNA gt IV Eluate highly pure plasmid DNA EcoRI Digestion linearized form of plasmid rE O MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification Table 4 Information about antibiotics according to Maniatis Antibiotic Stock solution Storage Working concenitra
27. lysate onto the corresponding NucleoBond AX column and discard the bottle top filter Figure 3 Correct use of the Nu cleoBond bottle top filter MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification 3 6 Elution procedures 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 20 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 20 25 C isopropanol to prevent spontaneous co precipitation of salt MACHEREY NAGEL 03 2005 Rev 03 15 Plasmid DNA Purification 4 Storage conditions and preparation of working solutions Attention Buffer S2 contains sodium dodecylsulfate and sodium hydroxide Wear gloves and goggles All kit components can be stored at room temperature 20 25 C and are sta ble up to two years Before you start any NucleoBond Plasmid DNA purification prepare the following e Dissolve the lyophilized RNase A by the addition of 1 ml of buffer S1 Wearing gloves is recommended Pipette up and down until the RNase A is dissolved completely Transfer the RNase A solution back to the bottle containing buffer S1 and shake well Indicate date of RNase A addition The final concentration of RNase A is 100 ug ml buffer S1 Store buffer S1 wit
28. nation it is sufficient for NucleoBond AX 500 columns Cat No 740531 5 preparations high copy plasmid purification e NucleoBond AX 100 columns Cat No 740521 10 preparations high copy plasmid purification NucleoBond AX 20 columns Cat No 740511 50 preparations high copy plasmid purification The NucleoBond BAC 100 kit is recommended for the isolation of low copy plas mids and contains sufficient buffer to perform 10 maxi preps The kit contains BAC 100 columns which can bind up to 500 ug of plasmid DNA Typically yields are 10 100 ug from 500 ml fermentation broth depending on copy number and size of con structs also see section 6 for further information regarding the growing of bacterial cultures The protocol for the isolation of low copy plasmids using the NucleoBond BAC 100 kit can be found in section 7 5 MACHEREY NAGEL 03 2005 Rev 03 13 Plasmid DNA Purification 3 5 Filtration of the lysate After alkaline lysis the solution has to be clarified from e g the cell debris through the supplied NucleoBond folded filters or NucleoBond bottle top filters in order to prevent clogging of the column Use the provided 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 fil ters completely remove SDS and cellular debris from plasmid samples Furthermore Folded Fil
29. nsion is EA Add pre cooled buffer S3 4 C to the suspension Immediately mix the lysate gently by inverting the flask 6 8 times until a homogeneous suspension contain ing an off white flocculate is formed Incubate the suspension on ice for 5 min ar Hem MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification 7 KI AX 20 AX 100 AX 500 Equilibration of the column Equilibrate a NucleoBond AX 20 Mini AX 100 Midi or AX 500 Maxi column with buffer N2 Allow the column to empiy by gravity flow Discard flow through sm EEI Clarification of the lysate Clear the bacterial lysate by following EITHER option 1 Midi Maxi or option 2 Mini Midi Maxi described below This step is extremely important excess precipitate left in suspension may clog the NucleoBond column in later steps Option 1 Filter the suspension Place a NucleoBond folded filter in a small funnel for support and prewet the filter with a few drops of buffer N2 or sterile deionized H O Load the bacterial lysate onto the wet filter and collect the flow through Note Do not use NucleoBond folded filters with AX 20 columns Mini preps Alternatively Option 2 Centrifuge the suspension Centrifuge at gt 12 000 x g for the minimum time indicated below at 4 C If the suspension contains residual precipitate after the first centrifugation either repeat this step or proceed with option 1 25 min K Binding Load the cleared
30. o lyse strains Isolate plasmid DNA from difficult to lyse strains by first resuspending the pellet in buffer S1 containing lysozyme 2 mg ml final concentration Incubate at 37 C for 30 minutes then continue with the addition of buffer S2 and proceed with the appro priate NucleoBond protocol MACHEREY NAGEL 03 2005 Rev 03 19 Plasmid DNA Purification 7 7 1 NucleoBond plasmid purification General procedure Prepare an overnight culture Set up an overnight bacterial culture by inoculating the appropriate volume of LB medium plus antibiotic with a single colony picked from a freshly streaked plate Shake the culture overnight 12 16 h with selecting antibiotics added to the medium Centrifuge the culture at 6 000 x g for 15 min at 4 C Carefully discard the supernatant 7 2 High copy plasmid purification Mini Midi Maxi Na AX 20 AX 100 AX 500 1 Cultivate and harvest bacterial cells 20 Harvest bacteria from an LB culture by centrifugation at 4 500 6 000 x g for 15 min at 4 C Cell lysis Carefully resuspend the pellet of bacterial cells in buffer S1 RNase A Please see section 6 3 regarding difficult to lyse strains za En Add buffer S2 to the suspension Mix gently by inverting the tube 6 8 times In cubate the mixture at room temperature 20 25 C for 2 3 min max 5 min Do not vortex as this will release contaminating chromosomal DNA from the cellular debris into the suspe
31. osed vials on ice for some hours In this case the eluate should be prewarmed to room temperature before the plasmid DNA is precipitated 15 ml 25 ml Optional Determine plasmid yield by UV spectrophotometry in order to adjust the de sired concentration of DNA step 10 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Mix carefully and centrifuge at 15 000 x g for 30 min at 4 C Carefully discard the supernatant Wash and dry DNA pellet Add room temperature 70 ethanol to the pellet Vortex briefly and centrifuge at 15 000 x g for 10 min at room temperature 20 25 C 7 ml f Carefully remove ethanol from the tube with a pipette tip Allow the pellet to dry at room temperature 20 25 C Drying for longer periods will not harm the quality of the plasmid DNA Reconstitute DNA Redissolve the DNA pellet in an appropriate volume of buffer TE or sterile deionized H O Depending on the type of centrifugation tube redissolve under constant spinning in a sufficient amount of buffer for 10 60 min 3D shaker Determine plasmid yields by UV spectrophotometry Confirm plasmid integrity by agarose gel electrophoresis MACHEREY NAGEL 03 2005 Rev 03 31 Plasmid DNA Purification 8 Appendix 8 1 Determination of DNA yield and quality Plasmid yield is measured by UV spectroscopy using the following relation Di at 260 nm 1 cm path length is equivalent to 50 ug plasmid
32. other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole MACHEREY NAGEL 03 2005 Rev 03 39 Plasmid DNA Purification 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 an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in ship ping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product against defects in products or components not manufactured by MACHEREY NAGEL or against damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPE CIFICALLY 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 RE PRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RE SPECT 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
33. s of any of those applications Please contact MACHEREY NAGEL Germany Tel 49 2421 969 270 and 275 e mail TECH BlIO mn net com 40 MACHEREY NAGEL 03 2005 Rev 03
34. the eluate can be stored in closed vials on ice for some hours In this case the eluate should be prewarmed to room temperature before the plasmid DNA is precipitated Optional Determine plasmid yield by UV spectrophotometry in order to adjust the de sired concentration of DNA step 10 MACHEREY NAGEL 03 2005 Rev 03 27 Plasmid DNA Purification Midi AX 20 AX 100 10 28 Precipitation Add room temperature isopropanol to precipitate the eluted plasmid DNA Cen trifuge at 15 000 x g for 30 min at 4 C Carefully discard the supernatant 0 75 ml 3 5 ml Wash and dry DNA pellet Add room temperature 70 ethanol to the pellet Vortex briefly and centrifuge at 15 000 x g for 10 min at room temperature 20 25 C 2 ml Carefully remove ethanol from the tube with a pipette tip Allow the pellet to dry at room temperature 20 25 C Drying for longer periods will not harm the quality of the plasmid DNA 5 10 min Reconstitute DNA Redissolve the DNA pellet in an appropriate volume of buffer TE or sterile deionized H O Depending on the type of centrifugation tube redissolve under constant spinning in a sufficient amount of buffer for 10 60 min 3D shaker Determine plasmid yields by UV spectrophotometry Confirm plasmid integrity by agarose gel electrophoresis MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification 7 5 Low copy plasmid purification Maxi BAC Mega Mega AX 500 BAC 100
35. tion concenitration Ampicillin 50 mg ml in H O 20 C 20 60 ug ml Chloramphenicol 34 mg ml in EtOH 20 C 25 170 ug ml Kanamycin 10 mg ml in H O 20 C 10 50 ug ml Streptomycin 10 mg ml in H O 20 C 10 50 ug ml Tetracycline 5 mg ml in EtOH 20 C 10 50 pg ml Maniatis T Fritsh EF Sambrook J Molecular cloning A laboratory manual Cold Spring Harbour Cold Spring New York 1982 34 MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification Problem Possible cause and suggestions SDS or other precipitates are present in the sample e Load the S1 2 3 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 or centrifugation but if the cleared lysate is stored on ice for a longer period new precipitates may appear If precipitate is visi ble it is recommended to filter respectively centrifuge the lysate again immediately before loading it onto the NucleoBond col umn pH or salt concentrations of buffers are too high e Especially if the customer prepares additional buffer it is recom mended to thoroughly check the pH of each buffer Adjust pH or prepare new buffers if necessary Sample lysate is too viscous Watch maximal volumes and pellet wet weights given in the manual Otherwise filtration of the lysate and flow rate of the cartridge will be insufficient No or low Column
36. ty flow Discard flow through 2 5 ml l MACHEREY NAGEL 03 2005 Rev 03 Plasmid DNA Purification AX 20 AX 100 Clarification of the lysate Clear the lysate by following EITHER option 1 or option 2 described below This step is extremely important excess precipitate left in suspension may clog the NucleoBond column in later steps Option 1 Filter the suspension Place a NucleoBond folded filter in a small funnel for support and prewet the filter with a few drops of buffer N2 or sterile deionized H O Load the lysate onto the wet filter and collect the flow through Note Do not use NucleoBond folded filters with AX 20 columns Mini preps Alternative Option 2 Centrifuge the suspension Centrifuge at gt 12 000 x g for the minimum time indicated below at 4 C If the suspension contains residual precipitate after the first centrifugation either repeat this step or proceed with option 1 Binding Load the cleared lysate from step 4 onto the NucleoBond column Allow the col umn to empty by gravity flow You may want to save all or part of the flow through for analysis Washing Wash the column with buffer N3 Repeat as indicated Discard flow through Elution Elute the plasmid DNA with buffer N5 Preheating buffer N5 to 50 C prior to elu tion may improve yields for high molecular weight constructs such as BACs We recommend precipitating the eluate as soon as possible step 8 Neverthe less
37. uffer N3 Buffer N3 Buffer N3 Washing high copy high copy high copy high copy high copy 2x1 5ml 10 ml 32 ml 2x35 ml 2x 100 ml low copy low copy low copy low copy 2x2 mi 12 ml 2x18ml 2x50 ml Elution Buffer N5 Buffer N5 Buffer N5 Buffer N5 Buffer N5 1 ml 5 ml 15 ml 25 ml 100 ml 3 Precipitation Isopropanol Isopropanol Isopropanol Isopropanol Isopropanol 0 75 ml 3 5 ml 11 ml 18 ml 70 ml 15 000 xg gt 15 000 xg gt 15 000 xg gt 15 000 xg gt 15 000 x g 30 min at 4 C 30 min at 4 C 30 min at 4 C 30 min at 4 C 30 min at 4 C gt lt Wash and dry 70 ethanol 70 ethanol 70 ethanol 70 ethanol 70 ethanol DNA pellet 500 jil 2 ml 5 ml 7 ml 10 ml gt 15 000 x g gt 15 000 x g gt 15 000 x g gt 15 000 x g gt 15 000 x g J 10 min at RT 10 min at RT 10 min at RT 10 min at RT 10 min at RT 5 10 min 5 10 min 10 20 min 30 60 min 30 60 min i Reconstitute Appropriate Appropriate Appropriate Appropriate Appropriate DNA volume of TE volume of TE volume of TE volume of TE volume of TE Plasmid DNA Purification Table of contents 1 2 Kit contents Introduction 2 1 Properties 2 2 About this user manual Product description 3 1 The basic principle 3 2 Kit specifications 3 3 Buffer compositions 3 4 High low copy plasmid purification 3 5 Filtration of the lysate 3 6 Elution procedures Storage conditions and preparation of working solutions Safety instructions risk and safety phrases Growing of b
38. ute DNA Redissolve the DNA pellet in an appropriate volume of buffer TE or sterile deionized H O Depending on the type of centrifugation tube redissolve under constant spinning in a sufficient amount of buffer for 10 60 min 3D shaker Determine plasmid yields by UV spectrophotometry Confirm plasmid integrity by agarose gel electrophoresis MACHEREY NAGEL 03 2005 Rev 03 25 Plasmid DNA Purification 7 4 Low copy plasmid purification Mini Midi Midi AX 20 AX 100 26 Cultivate and harvest bacterial cells Harvest bacteria from an LB culture by centrifugation at 4 500 6 000 x g for 15 min at 4 C Cell lysis Carefully resuspend the pellet of bacterial cells in buffer S1 RNase A Please see section 6 3 regarding difficult to lyse strains 8 0 ml Add buffer S2 to the suspension Mix gently by inverting the tube 6 8 times In cubate the mixture at room temperature 20 25 C for 2 3 min max 5 min Do not vortex as this will release contaminating chromosomal DNA from the cellular debris into the suspension 8 0 ml Add pre cooled buffer S3 4 C to the suspension Immediately mix the lysate gently by inverting the flask 6 8 times until a homogeneous suspension contain ing an off white flocculate is formed Incubate the suspension on ice for 5 min 8 0 ml Equilibration of the column Equilibrate a NucleoBond AX 20 Mini AX 100 Midi column with buffer N2 Allow the column to empty by gravi
39. y controlled and monitored the beads are uniform in diame ter and contain particularly large pores These special properties allow for optimum flow rates through the column and more efficient binding of nucleic acids to the ma trix Thus using the matrix you can achieve sharp well defined elution profiles for individual nucleic acid species see Figure 1 NucleoBond AX can separate dis tinct nucleic acids from each other and from proteins carbohydrates and other un wanted cellular components The purified nucleic acid products are suitable for use in the most demanding molecular biology applications including transfection in vitro transcription automated or manual sequencing cloning hybridization and PCR Compound class Plasmid DNA DNA i Double stranded DNA 150 bp Single stranded DNA M 13 LE mRNA 16S 23S rRNA 5S RNA tRNA Bovine serum albumin trinucleotides buffer N2 buffer N3 Proteins dyes T e T polysaccharides metabolites 0 5 M 1M 1 5 M Salt concentration for elution Figure 1 Elution profiles for distinct nucleic acid species using NucleoBond AX columns Nucleic acids can be eluted over a range of 0 5 M KCI to 1 5 M KCI profiles for each nucleic acid are sharp and virtually non overlapping MACHEREY NAGEL 03 2005 Rev 03 9 Plasmid DNA Purification 2 2 About this user manual Experienced users who are performing the purification of high copy plasmids using a Nu
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