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www.mn-net.comMACHEREY-NAGEL GmbH & Co. KG · Neumann-Neander-Str. 6–8 · 52355 Düren · GermanyFrance:MACHEREY-NAGEL EURLTel.: +33 388 68 22 68Fax: +33 388 51 76 88E-mail: [email protected]
Switzerland:MACHEREY-NAGEL AGTel.: +41 62 388 55 00Fax: +41 62 388 55 05E-mail: [email protected]
Germanyand international:Tel.: +49 24 21 969-0Fax: +49 24 21 969-199E-mail: [email protected]
MACHEREY-NAGEL
EN ISO 9001: 2008CERTIFIED
USA:MACHEREY-NAGEL Inc.Tel.: +1 484 821 0984Fax: +1 484 821 1272E-mail: [email protected]
Plas
mid
Pur
ifica
tion
Gui
de Plasmid purification productsfrom MACHEREY-NAGEL
MN guide to plasmid purification Find the optimal solutionNucleoBond®
NucleoSpin®
Superior yieldsOutstanding puritiesTime-saving procedures… for reliable downstream applications
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NucleoSpin®
Silica-membrane technology .......................................NucleoSpin® kits see page 6DNAcontaminants
Sample lysis, release of DNA from cells, tissue
DNA is bound to the silica membrane under high-salt conditions Interaction between DNA (hydrate shell is reversibly removed by chaotropic salt) and silica membrane
Contaminants are washed away under high-salt and / or ethanolic conditions to keep the DNA bound to themembrane
DNA is eluted in low-salt buffer or water, DNA is ready to use for downstream applications
Principle of binding: Removing of hydrate shell Hydrogen bonds / Salt bridges
Principle of elution: Reconstitution of hydrate shell
Features / Results •SequencingandPCR-gradeplasmidDNA •Noalcoholprecipitationnecessary •Fastandeasyprocedure
MN technologies for plasmid purificationNucleoBond® Anion-exchange technology ........................................NucleoBond® kits see page 3
DNAcontaminants
DNA is bound to the anion-exchanger matrix under low-pH conditions Interaction between positively charged anion-exchanger group and negatively charged DNA backbone
Stringent washing with increasing salt concentration to remove contaminants
DNA is eluted with high-pH buffer
Desalting / Concentration: Alcohol precipitation of eluted DNA DNA is collected by centrifugation or by using the NucleoBond®Finalizer
Si spacer NH
CH3
OHO
CH2
O
P
O
O
O
anion-exchangergroup MAE
DNA backbone
binding
Principle of binding: Ionic bond
2
3
O
CH
O
P
O
O
O
elution of DNA
anion-exchangergroup MAE
DNA backbone
pH shift
Si spacer N
CH
OH
Principle of elution: pH shift
Features / Results •Ultra-pure,transfection-gradeplasmidDNA •Thenewgenerationofanionexchangers •Xtrafast,Xtrahighyield,Xtraconvenient
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JJNucleoBond® Xtra Midi • NucleoBond® Xtra Maxi Superior plasmid Midi and Maxi kits – transfection-gradeXX Highest speed – 30 min (Midi) / 35 min (Maxi) Optimalcolumndesignandinsertedfilters–avoidinconvenientsyringesXX Highest yield – LyseControl for visualization of completed alkaline lysis Typically250μgforMidiand1000μgforMaxiXX High purity – transfection-grade plasmid DNA
Established anion-exchange technologyXX NucleoBond ® Xtra Plus kits for super high-speed version
NucleoBond®Finalizerforomittingtime-consumingcentrifugationafterDNAprecipitation
Product at a glanceSample material Midi
<200mL(highcopy)<400mL(lowcopy)
Maxi<600mL(highcopy)<1200mL(lowcopy)
Vectorsize <300kbp <300kbpTypicalyield 250μg 1000μgPrecipitation Midi Midi Plus
Centrifugation NucleoBond®FinalizerMaxi Maxi PlusCentrifugation NucleoBond® Finalizer
Preparation time 70min/prep 30min/prep 75min/prep 35min/prepApplications Transfection,cloning,plasmidbanks,etc. Transfection,cloning,plasmidbanks,etc.
Application data
resuspension, lysisneutralization, extra incubation
lysate clearing extra step (filtration, centrifugation)purification (equilibration, loading, washing, elution) plasmid yield
precipitation, dissolving
Preparation time [min]0 20 40 60 80 100 120 140 160 180 200
competitor Q750 μg
380 μg competitor I
1230 μg NucleoBond®
Xtra MaxiB
795 μg competitor Q
(HiSpeed)
1115 μg * NucleoBond®
Xtra Maxi PlusA
Maxi
Preparation time [min]0 20 40 60 80 100 120 140 160 180 200
260 μg competitor Q
125 μg competitor I
580 μg NucleoBond®
Xtra MidiB
520 μg * NucleoBond®
Xtra Midi Plus
335 μg competitor Q
(HiSpeed)
A
Midi
* Yield of plasmid DNA is slightly lower due to residual DNA remaining on the desalting tool (compared to kits without desalting tool).
Highest yield in less time – comparison to anion-exchange competitor kits
PlasmidDNAwas isolated followingeachmanufacturer’sprotocolusingmaximumculturevolume.PlasmidDNAyield () was determined after precipitation.ThecomparisonshowstheresultsofthekitswithNucleoBond®Finalizerasdesaltingtool(Fig.A)andwithoutdesaltingtool(Fig.B).
Customer testimonial„We have tested many of the market leaders in anion-exchange purification, and the MACHEREY-NAGEL NucleoBond Xtra® kits excel in ease of use, speed of preparation, yield, and DNA quality.“EnriqueSaez,PhD,TheGenomicsInstituteofNovartisResearchFoundation,SanDiego,CA,USA
Ordering informationProduct Preps REF
NucleoBond ® Xtra Midi 10/50/100 740410.10/.50/.100
NucleoBond ® Xtra Midi Plus (incl. Finalizer) 10/50 740412.10/.50
NucleoBond ® Xtra Maxi 10/50/100 740414.10/.50/.100
NucleoBond ® Xtra Maxi Plus (incl. Finalizer) 10/50 740416.10/.50
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JJNucleoBond® Xtra Midi EF • NucleoBond® Xtra Maxi EF Superior plasmid kit for transfection of sensitive cell lines – endotoxin-freeXX Highest purity – endotoxin-free transfection of sensitive cell lines
Patented endotoxin removalXX Highest speed – 45 min (Midi) / 50 min (Maxi)
No extra incubation for endotoxin removal Optimalcolumndesignandinsertedfilters–avoidinconvenientsyringesXX Highest yield – typically 250 μg for Midi and 1000 μg for Maxi Improvedsilicamaterial–patenteddesign!XX NucleoBond ® Xtra Plus EF kits for super high speed version
NucleoBond®Finalizerforomittingtime-consumingcentrifugationafterDNAprecipitation
Product at a glanceSample material Midi EF
<200mL(highcopy)<400mL(lowcopy)
Maxi EF<600mL(highcopy)<1200mL(lowcopy)
Vectorsize <300kbp <300kbpTypicalyield 250μg 1000μgPrecipitation Midi EF Midi Plus EF
Centrifugation NucleoBond®FinalizerMaxi EF Maxi Plus EFCentrifugation NucleoBond® Finalizer
Preparation time 85min/prep 45min/prep 90min/prep 50min/prepApplications Transfection,cloning,plasmidbanks,etc. Transfection,cloning,plasmidbanks,etc.
Application dataTransfection of PC12 cells with plasmid DNA purified using NucleoBond® Xtra EF
Plasmid purification: 9.2 kbpplasmid,bacterial strain:E. coliDH5α,purificationwithNucleoBond®XtraMidiEF.
Transfection experiment:HighlysensitivePC12cells, in vitro cultivation, transfection with0.5μgplasmidDNA,transfectionmethod:Lipofectamine™LTXReagent(1μL)+100μLDMEM,cellswereimagedbyfluorescencemicroscopy24hpost-transfection.
Results:CellsshowtransientexpressionofPhogrin(PTPNP,ProteinTyrosinePhos-phatase), localizedbyredfluorescentantibody,nucleiare localizedbyDAPIstaining(violet).
DatakindlyprovidedbyAGKnauer,Duisburg-EssenUniversity,InstituteforMolecularBiologyII,Essen,Germany
Customer testimonial„Our lab is constantly doing transfections and we use no other kit but the MACHEREY-NAGEL NucleoBond Xtra® Maxi EF. It is easy and fast to use and always gives us great, consistent results so that we spend less time prepping plasmids and more time on experiments that matter.“ MollyRomine,Pediatrics,WashingtonUniversitySchoolofMedicine,St.Louis,MO,USA
Ordering informationProduct Preps REF
NucleoBond ® Xtra Midi EF 10/50 740420.10/.50
NucleoBond ® Xtra Midi Plus EF (incl. Finalizer) 10/50 740422.10/.50
NucleoBond ® Xtra Maxi EF 10/50 740424.10/.50
NucleoBond ® Xtra Maxi Plus EF (incl. Finalizer) 10/50 740426.10/.50
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JJNucleoBond® Xtra BAC Kit for large construct DNA XX High yield – up to 150 μg BAC DNA
Optimal silica material for large constructs LyseControlforvisualizationofefficientalkalinelysisXX High speed – 75 minutes per prep OptimalcolumndesignandinsertedfiltersforhighflowratesandparallellysateclearingandloadingXX High purity – transfection-grade BAC DNA
Established anion-exchange technology
Product at a glanceSample material 250–750mLE. coli cultureVectorsize <300kbpTypicalyield 10–150μgPreparation time 75min/prepApplications Transfection,cloning,restrictionanalyses,sequencing
Application datSuperior yield in less time – comparison to competitor kits
BACDNA (300kbp)was isolated in triplicate from500mLE. coliDH5αusingNucleoBond®XtraBACandcompetitorproducts(QandI).Afterprecipitation,BACDNAwasreconstitutedin1000μLTEbufferand5μLofeachsamplewasusedforanalysisona1%TAE-agarosegel.Obtained yields:MN:150μg,Q:44μg,I:75μg
Ordering informationProduct Preps REF
NucleoBond ® Xtra BAC 10/25 740436.10/.25
JJNucleoBond® 96 Xtra EF High-throughput isolation of endotoxin-free plasmid DNAXX Highest purity – endotoxin-free transfection of sensitive cell lines
Patented endotoxin removalXX NucleoBond® Filter Plate for convenient filtration of bacterial lysates
Processing under vacuum or by centrifugation InnovativeMNWashPlateminimizesriskofcross-contaminationXX NucleoBond® Finalizer Plate to speed up DNA precipitation
Product at a glanceSample material 1–5mLE. coli cultureVectorsize <15kbp
<300kbp(withoutNucleoBond®FinalizerPlate)Typicalyield 2–4μg(1.5mLLB/TBin96-wellcultureplates)
10–50μg(5mLLB/TBinglasstubes)Preparation time 120min/plateApplications Transfection,cloning,plasmidbanks,etc.
Ordering informationProduct Preps REF
NucleoBond ® 96 Xtra EF 1x96/4x96 740430.1/.4
MN Q I
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JJNucleoSpin® Plasmid · NucleoSpin® Plasmid (NoLid) High-yield plasmid Mini prep – molecular biology- / sequencing-grade XX Highest yield Upto50μgplasmidDNAXX Highest quality Phredscoreof20(upto1000bp)XX Fast procedure 6Miniprepsin25minXX Choose between columns with or without a lid
Product at a glanceSample material 1–5mLE. coli cultures (standard protocol)
5–10mLE. coli cultures* Vectorsize <15kbpTypicalyield <25μg(from1–5mL)
<50μg(from5–10mL)*Elution volume 50μLBinding capacity 60μgApplications Sequencing,cloning,PCR,restrictionanalysis,transformation,enzymaticmodifications
Application data M 1 2 3 4 5 6 High-yield plasmid Mini prep
PlasmidDNAisolation(pUC18)fromE. coliDH5αusingNucleoSpin®Plasmid.For the isolation2mL (lane1–2),5mL (lane3–4)and8mL (lane5–6)LBcultureswereusedandanalyzedonanagarosegel (2μLof eacheluate). LaneM:marker.
Ordering informationProduct Preps REF
NucleoSpin® Plasmid 10/50/250 740588.10/.50/.250
NucleoSpin® Plasmid (NoLid) 50/250 740499.50/.250
*Iflargervolumesarefrequentlyused,increasedbuffervolumesarerequired.Pleasesee“orderinginformation”(page8).
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JJNucleoSpin® Plasmid EasyPure High-speed plasmid Mini prep – molecular biology- / sequencing-gradeXX Highest speed – one combined washing and drying step 6Miniprepsin14minXX LyseControl for visualization of completed alkaline lysis Obtainupto35μgplasmidDNAXX Now with Liquid RNase
Easy handling without dissolving
Product at a glanceSample material 2–10mLE. coli cultures Vectorsize <15kbpTypicalyield 15–30μgElution volume 50μLBinding capacity 35μg
Ordering informationProduct Preps REF
NucleoSpin® Plasmid EasyPure 10/50/250 740727.10/.50/.250
JJNucleoSpin® 8 / 96 Plasmid · NucleoSpin® 8 / 96 Plasmid Core Kit High-throughput plasmid Mini prep – molecular biology- / sequencing-gradeXX Convenient filtration of bacterial lysates
NucleoSpin®8FilterStrips/96FilterPlate OptimizedfiltermaterialminimizestheriskoffiltercloggingXX Minimized risk of cross-contamination MNWashPlateXX Processing under vacuum or by centrifugation*
Suitable for manual and automated processingXX NucleoSpin® 8 / 96 Plasmid Core Kit: Kitswithbasiccontentfocusedonautomationplatforms–additionalaccessoriescanbecombinedasneeded
Product at a glanceSample material 1–5mLE. coli cultureVectorsize <15kbpTypicalyield 4–6μg/mLE. coli cultureElution volume 75–150μLPreparation time 45min / 6stripsor1plateBinding capacity 20μgApplications Sequencing,cloning,PCR,restrictionanalysis,transformation,enzymaticmodifications
Ordering informationProduct Preps REF
NucleoSpin® 8 Plasmid 12x8/60x8 740621/.5
NucleoSpin® 8 Plasmid Core Kit 48 x 8 740461.4
NucleoSpin® 96 Plasmid 1x96/4x96/24x96 740625.1/.4/.24
NucleoSpin® 96 Plasmid Core Kit 4x96/24x96 740616.4/.24
*SupportprotocolavailableatTechnicalSupport([email protected])
Your local distributor
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www.mn-net.comMACHEREY-NAGEL GmbH & Co. KG · Neumann-Neander-Str. 6–8 · 52355 Düren · GermanyFrance:MACHEREY-NAGEL EURLTel.: +33 388 68 22 68Fax: +33 388 51 76 88E-mail: [email protected]
Switzerland:MACHEREY-NAGEL AGTel.: +41 62 388 55 00Fax: +41 62 388 55 05E-mail: [email protected]
Germanyand international:Tel.: +49 24 21 969-0Fax: +49 24 21 969-199E-mail: [email protected]
MACHEREY-NAGEL
EN ISO 9001: 2008CERTIFIED
USA:MACHEREY-NAGEL Inc.Tel.: +1 484 821 0984Fax: +1 484 821 1272E-mail: [email protected]
Ordering informationTransfection-grade plasmid DNA purification kits Preps REF
NucleoBond® Xtra Midi 10/50/100 740410.10/.50/.100
NucleoBond® Xtra Midi Plus (incl. Finalizer) 10/50 740412.10/.50
NucleoBond® Xtra Maxi 10/50/100 740414.10/.50/.100
NucleoBond® Xtra Maxi Plus (incl. Finalizer) 10/50 740416.10/.50
NucleoBond® Xtra BAC 10/25 740436.10/.25
Transfection-grade, endotoxin-free plasmid DNA purification kits Preps REF
NucleoBond® Xtra Midi EF 10/50 740420.10/.50
NucleoBond® Xtra Midi Plus EF (incl. Finalizer) 10/50 740422.10/.50
NucleoBond® Xtra Maxi EF 10/50 740424.10/.50
NucleoBond® Xtra Maxi Plus EF (incl. Finalizer) 10/50 740426.10/.50
NucleoBond® 96 Xtra EF 1x96/4x96 740430.1/.4
Molecular biology-/sequencing-grade plasmid DNA purification kits Preps REF
NucleoSpin® Plasmid 10/50/250 740588.10/.50/.250
NucleoSpin® Plasmid (NoLid) 50/250 740499.50/.250
NucleoSpin® Plasmid EasyPure 10/50/250 740727.10/.50/.250
NucleoSpin® 8 Plasmid 12x8/60x8 740621/.5
NucleoSpin® 8 Plasmid Core Kit 48 x 8 740461.4
NucleoSpin® 96 Plasmid 1x96/4x96/24x96 740625.1/.4/.24
NucleoSpin® 96 Plasmid Core Kit 4x96/24x96 740616.4/.24
Related productsBuffer sets Pack of REF
NucleoBond® Xtra Buffer Set I 1 740417
NucleoBond® Xtra BAC Buffer Set 1 740437
NucleoBond® Xtra EF Buffer Set I 1 740427
NucleoSpin® Plasmid Buffer Set 1 740953
Product accessories Pack of REF
NucleoBond® Xtra Combi Rack 1 740415
NucleoVac Vacuum Manifold 1 740681
NucleoVac Vacuum Regulator 1 740641
Trademarks MACHEREY-NAGEL: NucleoSpin®, NucleoBond® Other companies: Lipofectamin™[Invitrogen]
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idGuideen3/10/0/08.2013PD
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