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MACHEREY-NAGEL
EN ISO 9001EN ISO 13485
CERTIFIED
MACHEREY-NAGEL GmbH & Co. KG · Neumann-Neander-Str. 6–8 · 52355 Düren · GermanyFrance:MACHEREY-NAGEL EURLTel.: +33 388 68 22 68E-mail: [email protected]
Switzerland:MACHEREY-NAGEL AGTel.: +41 62 388 55 00E-mail: [email protected]
Germanyand international:Tel.: +49 24 21 969-0E-mail: [email protected]
USA:MACHEREY-NAGEL Inc.Tel.: +1 484 821 0984E-mail: [email protected]
Large construct DNA purification
User manualNucleoBond® Xtra BAC
March 2014 / Rev. 02
A041
320
/064
0.6
Large construct DNA purification (NucleoBond® Xtra BAC)Protocol-at-a-glance (Rev. 02)
MACHEREY-NAGEL GmbH & Co. KG · Neumann-Neander-Str. 6–8 · 52355 Düren · Germany Tel.: +49 24 21 969-270 · Fax: +49 24 21 969-199 · [email protected] · www.mn-net.com
1 Prepare a starter culture
2 Prepare a large overnight culture
3 Harvest bacterial cells 4,500–6,000 x g
≥ 10 min at 4 °C
4 Resuspension 60 mL Buffer RES-BAC
5 Cell lysis(Important: Check Buffer LYS-BAC for precipitated SDS)
60 mL Buffer LYS-BAC
Invert the tube 5 times
RT, 5 min
6 Equilibration of the column and filter
30 mL Buffer EQU-BAC
7 Neutralization 60 mL Buffer NEU-BAC
Invert the tube 10–15 times, until sample turnes colorless
0 °C / on ice, ≥ 5 min
8 Clarification and loading of the lysate
Invert the tube 3 times
Load lysate on NucleoBond® Xtra BAC Column Filter
9 Wash column filter and column 15 mL Buffer EQU-BAC
10 Discard column filter Discard NucleoBond® Xtra BAC Column Filter
11 Wash column 45 mL Buffer WASH-BAC
12 Elution 15 mL Buffer ELU-BAC
65–70 °C
13 Precipitation 6 mL isopropanol
RT, 2 min
15,000 x g 30 min at 4 °C
14 Wash and dry DNA pellet 5 mL 70 % ethanol
15,000 x g 5 min at RT
10–15 min
15 Reconstitute DNA Appropriate volume of TE buffer or sterile H2O (pH ≥ 7)
3
Large construct DNA purification
MACHEREY-NAGEL – 03 / 2014, Rev. 02
Table of contents
1 Components 41.1 Kit contents 41.2 Reagents and equipment to be supplied by user 5
2 Kitspecifications 6
3 About this user manual 7
4 NucleoBond®XtraBACplasmidpurificationsystem 84.1 Basicprinciple 84.2 NucleoBond®XtraBACanion-exchangecolumns 84.3 Growth of bacterial cultures 104.4 Chloramphenicolamplificationoflow-copyplasmids 114.5 Culture volume for large constructs 124.6 LysateneutralizationandLyseControl 124.7 Cell lysis 134.8 Difficult-to-lysestrains 144.9 Setup of NucleoBond® Xtra BAC Columns 144.10 Filtration and loading of the lysate 154.11 Washing of the column 154.12ElutionoflargeconstructDNA 164.13ConcentrationoflargeconstructDNA 164.14DeterminationofDNAyieldandquality 164.15 Convenient stopping points 17
5 Storageconditionsandpreparationofworkingsolutions 18
6 Safetyinstructions 19
7 NucleoBond®XtraBACpurification 21
8 Appendix 268.1 Troubleshooting 268.2 Orderinginformation 338.3 Productuserestriction/warranty 34
Large construct DNA purification
MACHEREY-NAGEL – 03 / 2014, Rev. 024
1 Components
1.1 Kit contents
NucleoBond® Xtra BAC
10 preps 25 prepsREF 740436.10 740436.25
Buffer RES-BAC 750mL 2x1000mL
BufferLYS-BAC 750mL 2x1000mL
Buffer NEU-BAC 750mL 2x1000mL
Buffer EQU-BAC 500mL 1000mL 500mL
Buffer WASH-BAC 500mL 1000mL 250mL
BufferELU-BAC 200mL 500mL
RNaseA*(lyophilized) 75 mg 2 x 100 mg
NucleoBond® Xtra BAC Columns incl. NucleoBond® Xtra BAC Column Filters
10 25
PlasticWashers 5 10
User manual 1 1
* For preparation of working solutions and storage conditions see section 5.
5
Large construct DNA purification
MACHEREY-NAGEL – 03 / 2014, Rev. 02
1.2 Reagents and equipment to be supplied by user
Reagents
• Isopropanol(room-temperatured)• 70%ethanol(room-temperatured)• BufferforreconstitutionofDNA,forexample,TEbufferorsterileH2O(pH≥7)
Equipment
• Standard microbiological equipment for growing and harvesting bacteria (e.g., inoculatingloop,culturetubesandflasks,37°Cshakingincubator,andcentrifugewithrotorandtubesorbottlesforharvestingcells)
• Refrigerated centrifuge capable of reaching ≥ 5,000xg with rotor for the appropriate centrifuge tubes or bottles
• Centrifugationtubesorvesselswithsuitablecapacityforthevolumesspecifiedin the respective protocol
• NucleoBond®XtraCombiRack(seeorderinginformation)orequivalentholder
Large construct DNA purification
MACHEREY-NAGEL – 03 / 2014, Rev. 026
2 Kit specifications• NucleoBond® Xtra BAC isdesignedforultrafastpurificationofcosmidsand
verylargeconstructs(P1,BACs,PACs)rangingfrom3kbpupto300kbp.Forpreparation of working solutions and storage conditions see section 5.
• NucleoBond® Xtra BAC Columns are polypropylene columns containing NucleoBond® Xtra BAC Silica Resinpackedbetweentwoinertfilterelements.Thecolumnsofferabindingcapacityof200μgforverylargeconstructs.
• All NucleoBond® Xtra BAC Columns are resistant to organic solvents such asalcohol,chloroform,andphenolandarealsosuitableforbufferscontainingdenaturing agents like formamide, urea, or common detergents like TritonX-100orNP-40.
• NucleoBond® Xtra BAC Silica Resin can be used over a wide pH range (pH2.5–8.5),andcanremainincontactwithbuffersforseveralhourswithoutany change in its chromatographic properties.
• TheNucleoBond® Xtra BAC Column Filters are specially designed depth filters that fit into theNucleoBond® Xtra BAC Columns. These filters areinserted and ready-to-use in the NucleoBond® Xtra BAC Columns. Thisallowsforatime-saving,simultaneousclearingofbacteriallysateandloadingof cleared lysate onto the NucleoBond® Xtra BAC Column.Asaresult,thetime-consuming centrifugation step for lysate clearing is avoided.
• The NucleoBond® Xtra BAC Column Filters allow complete removal of precipitatewithoutclogging(evenwith large lysatevolumes),andalsoavoidshearingoflargeDNAconstructs,suchasPACsorBACs.
• LyseControl:TheLysisBufferLYS-BACcontainsabluepHindicatortoensurecompleteneutralizationformaximumyield.
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Large construct DNA purification
MACHEREY-NAGEL – 03 / 2014, Rev. 02
3 About this user manualSection 4 provides you with a detailed description of the NucleoBond® Xtra BAC purification system and important information about cell growth, cell lysis, and thesubsequentpurificationsteps.Inaddition,sections5and6informyouaboutstorage,bufferpreparation,andsafetyinstructions.
First-time users are strongly advised to read these chapters thoroughly before using thiskit.Experienceduserscandirectlyproceedwiththepurificationprotocol(section7)orjustusetheProtocol-at-a-glanceforaquickreference.
Eachproceduralstepinthepurificationprotocolisarrangedlikethefollowingexampletaken from section 7:
5 Cell lysis (Buffer LYS-BAC)
Check Lysis Buffer LYS-BAC for precipitated SDS prior to use. If a white precipitate is visible, warm the buffer for several minutes at 30–40°C untilprecipitate is completely dissolved.Then, proceed to cool the buffer down toroomtemperature(18–25°C).
Add Lysis Buffer LYS-BAC to the suspension.
Mix gently by inverting the tube 5 times. Do not vortex as this will shear and release contaminating chromosomal DNA from cellular debris into thesuspension.
Incubatethemixtureatroomtemperature(18–25°C)for5 min.
Warning: Prolonged exposure to alkaline conditions can irreversibly denatureanddegradelargeconstructDNAandliberatecontaminatingchromosomalDNAinto the lysate.
Note: Increase LYS-BAC buffer volume proportionally if more than the recommended cell mass is used (see section 4.7 for information on optimal cell lysis).
60 mL
Youwillfindvolumesorincubationtimesinthewhiteboxes.Thenameofthebuffer,aswellas incubationtimes,repeatsor importanthandlingsteps,areemphasized inbold type within the instructions. Additional notes or optional steps are printed in italic. Theexclamationpointmarksinformationandhintsthatareessentialforasuccessfulpreparation.
IntheexampleshownaboveyouareaskedtochecktheLysisBufferLYS-BACpriortouse and then to lyse the resuspended cell pellet in 60 mL of Buffer LYS-BAC. Follow the handling instructions exactly and note the given hints for protocol alterations.
Large construct DNA purification
MACHEREY-NAGEL – 03 / 2014, Rev. 028
4 NucleoBond® Xtra BAC plasmid purification system
4.1 Basic principle
ThebacterialcellsarelysedbyanoptimizedsetofnewlyformulatedbuffersbasedontheNaOH/SDSlysismethodofBirnboimandDoly*.
After equilibration of the NucleoBond® Xtra BAC Column together with the corresponding NucleoBond® Xtra BAC Column Filter,theentirelysateisloadedbygravityflowandsimultaneouslyclearedbythespeciallydesignedcolumnfilter.
PlasmidDNAisboundtotheNucleoBond® Xtra BAC Silica Resin.
Uponcompletionof thewashing step, theplasmidDNA is eluted, precipitated, andeasilydissolvedinanysuitablebuffer(e.g.,low-saltbufferorwater)forfurtheruse.
4.2 NucleoBond® Xtra BAC anion-exchange columns
NucleoBond® Xtra BAC is a patented silica-based anion-exchange resin developed byMACHEREY-NAGEL.It isdevelopedforroutineseparationofdifferentclassesofnucleicacidssuchasoligonucleotides,RNA,plasmids,andlargeconstructslikeP1,BAC,orPAC.
NucleoBond® Xtra BAC Silica Resin consists of hydrophilic, macroporous silicabeads functionalized with MAE (methyl-amino-ethanol). The dense coating of thisfunctional group provides a high positive charge density under acidic pH conditions. As aresult,thispermitsthenegativelychargedphosphatebackboneofplasmidDNAtobindwithhighspecificity(Figure1).
Si spacer NH
CH3
OHO
CH2
O
P
O
O
O
anion-exchangergroup MAE
DNA backbone
binding
Figure 1: Ionic interaction of the positively charged methyl-hydroxyethyl-amino group with the negative phosphate oxygen of the DNA backbone.
IncontrasttothewidelyusedDEAE(diethylaminoethyl)group,thehydroxygroupofmethyl-hydroxyethyl-amin can be involved in additional hydrogen bonding interactions withtheDNA.
*Birnboim,H.C.andDoly,J.,(1979)Nucl.AcidsRes.7,1513-1523
9
Large construct DNA purification
MACHEREY-NAGEL – 03 / 2014, Rev. 02
Duetoaspecializedmanufacturingprocess,thatisstrictlycontrolledandmonitored,the NucleoBond® Xtra BAC silica beads are uniform in diameter and contain particularlylargepores.Thesespecialpropertiesallowoptimizedflowratesandsharp,well-defined elution profiles.NucleoBond® Xtra BAC can separate distinct nucleic acidspeciesfromeachotheraswellasproteins,carbohydrates,andotherunwantedcellularcomponentsoverabroadrangeofsaltconcentrations(Figure2).
AllcontaminantsfromproteinstoRNAarewashedfromthecolumn.Then,thepositivechargeoftheresinisneutralizedbyapHshifttoslightlyalkalineconditionsandpureplasmidDNAiselutedinahigh-saltelutionbuffer.
Thepurifiednucleicacidproductsaresuitableforuseinthemostdemandingmolecularbiologyapplications,includingtransfection,in vitrotranscription,automatedormanualsequencing,cloning,hybridization,andPCR.
0 0.5 1 1.5
Salt concentration for elution [M (KCl)]
Abso
rban
ce a
t 260
nm
Com
poun
d cl
ass Single-stranded DNA,
Double-stranded DNA
mRNA, 16S/23S rRNA
5S rRNA
tRNA
Proteins, dyes, polysaccharides, metabolites, trinucleotides
tRN
A
rRN
A
Plas
mid
DN
A,la
rge
cons
truct
s
Plasmid DNA,large constructs
Figure 2: Elution profile of NucleoBond® Xtra BAC Silica Resin at pH 7.0 Themoreinteractionsanucleicacidcanformbetweenthephosphatebackboneand
the positively charged resin, the later it is elutedwith increasing salt concentration.LargenucleicacidscarrymorechargesthansmalleronesanddoublestrandedDNAcarries more than single stranded RNA.
Large construct DNA purification
MACHEREY-NAGEL – 03 / 2014, Rev. 0210
4.3 Growth of bacterial cultures
Yield and quality of large constructDNAhighly depend on several factors: type of culture mediaandantibiotics,thebacterialhoststrain,thetypeofDNA,size,andcopynumber,butalsoonthegrowth conditions.
Forlargeconstructs,likeBAC,PAC,P1,orcosmidDNA,LB(Luria-Bertani)mediumisstronglyrecommended.ThecellcultureshouldbegrowntoanOD600 of 1.5–2.0 to preventstarvationofthecellsanddegradationoflargeconstructs.Therefore,incubateat 32–37°C preferably 12–16 hours overnight. Use flasks of at least three or fourtimes the volume of the culture volume and shake at 120–250 rpm to provide a growth medium fully saturated with oxygen.
Alternatively to LB, rich media like 2xYT (Yeast/Tryptone), TB (Terrific Broth), orCircleGrow can be used. In this case, bacteria grow faster, reaching the stationaryphase much earlier than in LB medium (≤12 h), and reach higher cell masses.However,thisdoesnotnecessarilyyieldmoreDNA.OvergrowingaculturemightleadtoahigherpercentageofdeadorstarvingcellsandtheresultingBAC,PAC,P1,orcosmidDNAmightbepartiallydegradedorcontaminatedwithchromosomalDNA.Tofindtheoptimalcultureconditions,theculturemediumandincubationtimeshavetobeoptimizedforeachhoststrain/largeconstructcombinationindividually.
Cell cultures should be grown under antibiotic selection at all times to ensure large constructDNApropagation.Withoutthisselectivepressure,cellstendtolosealargeconstruct during cell division. Since bacteria grow much faster without the burden of anadditionalchromosome,theytakeovertheculturerapidlyandtheyieldgoesdownregardlessofthecellmass.Table1givesinformationonconcentrationsofcommonlyused antibiotics.
Table 1: Information about antibiotics according to Maniatis*
Antibiotic Stock solution (concentration)
Storage Working concentration
Ampicillin 50mg / mLinH2O -20°C 50–100μg / mL
Chloramphenicol 34mg / mLinEtOH -20°C 25–170μg / mL
Kanamycin 10mg / mLinH2O -20°C 10–50μg / mL
Streptomycin 10mg / mLinH2O -20°C 10–50μg / mL
Tetracycline 5mg / mLinEtOH -20°C 10–50μg / mL
Carbenicillin 50mg / mLinH2O -20°C 20–60μg / mL
* ManiatisT,FritschEF,SambrookJ:Molecularcloning.Alaboratorymanual,ColdSpringHarbor,ColdSpring,NewYork1982.
11MACHEREY-NAGEL – 03 / 2014, Rev. 02
LargeconstructDNApurification
TheE. coli host strainmostlyinfluencesthequalityofthelargeconstructDNA.StrainslikeDH5αorXL1-Blueusuallyproducehighqualitysuper-coiledDNA,whereasstrainslikeHB101,withhigh levelsofendonucleaseactivity,mightyield lowerquality largeconstructs, giving poor results in downstreamapplications like enzymatic restrictionor sequencing.When noticing problemswith host strains likeTop10,HB101, or itsderivativeslikeTG1,JM100,achangetoDH5αorXL1-Blueshouldbeconsidered.
Thetype of large construct,especiallythe size and the origin of replication (ori), hasacrucialinfluenceonDNAyield.Ingeneral,thelargertheconstructortheclonedinsertis,thelowertheexpectedDNAyieldisduetoalowercopynumber.CosmidsorBACs,forexample,aremaintainedatcopynumbers<20anddowntoonly1,whereasvectorsbasedon,forexample,pUC,pBluescript,orpGEM,canbepresentinseveralhundred copies per cell.
Thus,alltheabovementionedfactorsshouldbetakenintoconsiderationifaparticularDNAyieldhastobeachieved.Culturevolumeandlysisprocedureshavetobeadjustedaccordingly.Thepreptoprepvariationofyieldvariesmuchmorecomparedtohigh-copyplasmidpurification.
4.4 Chloramphenicol amplification of low-copy plasmids
TodramaticallyincreasethelowcopynumberofpMB1 / colE1derivedplasmids,growthecellculturetomidorlatelogphase(OD600≈0.6–2.0)underselectiveconditionswithanappropriateantibiotic.Then,add170μg/mLchloramphenicolandcontinuetoincubateforanadditional8–12hours.Chloramphenicolinhibitshostproteinsynthesisand as a result, prevents replication of the host chromosome. Plasmid replication,however,isindependentofnewlysynthesizedproteinsandcontinuesforseveralhoursuntil up to 2000–3000 copies per cell are accumulated*.
Alternatively,thecellculturecanbegrownwithonlypartialinhibitionofproteinsynthesisunder low chloramphenicol concentrations (10–20 μg/mL), resulting in a 5–10-foldgreateryieldofplasmidDNA**.
BothmethodsshowthepositivesideeffectofmuchlessgenomicDNAperplasmid,butthey obviously work only with plasmids that do not carry the chloramphenicol resistance gene.Furthermore,themethodisonlyeffectivewithlowcopynumberplasmidsunderstringentcontrol (e.g.,pBR322).Allmodernhighcopynumberplasmids (e.g.,pUC)are already under relaxed control due to mutations in the plasmid copy number control genes,resultinginaninsignificantadditionalincreasetotheircopynumber.
* ManiatisT,FritschEF,SambrookJ:Molecular cloning. A laboratory manual,ColdSpringHarbor,ColdSpring,NewYork1982.
**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. 0212
4.5 Culture volume for large constructs
Due to the influence of growth media (TB, CircleGrow, 2xYT), growth conditions(shaking,temperature,incubationtime)andhoststrainortypeofinsertetc.,thefinalamountofcells inabacterialculturecanvaryoverawiderange.Byruleof thumb,1 liter of E. coliculturewithanOD600of 1 consists of 1 x 1012 cells and yields about 1.5–1.8gcellwetweight.OvernightculturescontainingalargeconstructandgrowninLBmediumusuallyreachanOD600of3undervigorousshakinginflasks.TheexpectedDNAyieldforalargeconstructisapproximately30–40μgpergramcellwetweight.
Asaconsequence, it is important toadjust the cell mass rather than the culture volume for the best large construct purification results. However, since the cellmassorcellwetweightistedioustodetermine,itwasreplacedinthismanualbythemathematicalproductofopticaldensityat600nm(OD600)andculturevolume(Vol),twovariables that are much easier to measure.
ODV = OD600 x Vol [mL]
Please note that for a correctOD determination, theculture samples have to be diluted if the OD600 exceeds 0.5, allowing it to increase proportionally with cell mass. For a well grown E. coli culture, a 1:10dilutionwith fresh culturemedium isrecommended.ThemeasuredOD600 is then multiplied with the dilution factor 10 to result ina theoreticalOD600 value.ThisOD600 is used to determine the appropriate culturevolume.Table2showsrecommendedODVsalongwiththecorrespondingpairsofOD600 and culture volume that can be easily handled using the standard kit protocol lysisbuffervolumes.Forexample,iftheOD600 of your E. colicultureis2,use750mLculture for your BAC preparation
Table 2: Recommended culture volumes for large constructs
NucleoBond® Xtra BAC
Pellet wet
weight
Rec. ODV
= OD600 x Vol
Recommended culture volume for
OD600 =
2
OD600 =
3
OD600 =
4
OD600 =
5
OD600 =
6
2.8g 1500 750 mL 500 mL 375 mL 300 mL 250 mL
4.6 Lysate neutralization and LyseControl
PropermixingofthelysatewithNeutralizationBufferNEU-BACisofutmostimportanceforcompleteprecipitationofSDS,protein,andgenomicDNA.Incompleteneutralizationleadstoreducedyields,slowerflow-rates,andpotentialcloggingoftheNucleoBond®
XtraColumnFilter.However,releasedplasmidDNAandespeciallylargeBACDNA,isveryvulnerableatthispointandshakingtoomuchortoostronglywilldamagetheDNA.
LargeconstructDNApurification
13MACHEREY-NAGEL – 03 / 2014, Rev. 02
Therefore,do not vortex or shake but just invert the mixture very gentlyuntilafluffyoff-white precipitate has formed and the LyseControl has turned colorless throughout the lysate without any traces of blue color.
4.7 Cell lysis
Thebacterial cell pellet is resuspended inBufferRES-BACand lysedbya sodiumhydroxide/SDS treatment with Buffer LYS-BAC. Proteins, as well as chromosomaland large construct DNA, are denatured under these conditions. In addition, RNAisdegradedbyDNase-freeRNaseA.Large construct lysates have to be treated even more carefully than plasmid DNA lysates to avoid nicking and irreversible denaturation of the large constructs. As a consequence, shaking or vortexing of the lysate must be avoided. NeutralizationBufferNEU-BAC,containingpotassiumacetate, is thenaddedto the lysate,causingSDStoprecipitateasKDS(potassiumdodecylsulfate)andpulldownproteins,chromosomalDNA,andothercellulardebris.Thepotassiumacetatebufferalsoneutralizesthelysate.CircularDNAcanreverttoitsnative super-coiled structure and remains in solution.
TheNucleoBond® Xtra BAC buffer volumes given in the standard protocol are ad-justedtoensureoptimallysisforculturevolumesgiveninsection4.5,Table2.Usingtoomuchcellmaterialleadstoinefficientcelllysisandprecipitation,whichcanpoten-tiallyreducelargeconstructyieldandpurity.Therefore,lysisbuffervolumesshouldbeincreased when applying larger culture volumes.
By rule of thumb, calculate the necessary lysis buffer volumes for RES-BAC, LYS-BAC, and NEU-BAC as follows:
Vol. [mL] = Culture Volume [mL] x OD600 / 25
Note: This formula differs from the one for plasmid DNA to ensure maximum yield for large constructs.
Forexample,if500mLofabacterialculture(OD600=3,ODV=1500)istobelysed,theappropriatevolumesoflysisbuffersRES-BAC,LYS-BAC,andNEU-BACare60mLeach.Ifmorelysisbufferisneededthanisprovidedwiththekit,anadditionalbufferset includingbuffersRES-BAC,LYS-BAC,NEU-BAC,andRNaseAcanbeorderedseparately(seeorderinginformation).
If less than the recommended amount of cells is to be used (e.g., due to bad cellgrowthorlimitedculturevolumes),lesslysisbuffervolumescanbeusedthangiveninthestandardprotocol(60mLeach).Calculatethenecessaryamountaccordingtotheabovegivenformula.Notethattheyieldmightthenbesignificantlylowerthan200μg.
Byusingsufficientamountsoflysisbuffer,lysistimecanbelimitedto3–4minutesandshouldnotexceed5minutes.Prolongedexposuretoalkalineconditionscanirreversiblydenature and degrade especially large construct DNA and liberate contaminatingchromosomalDNAintothelysate.
LargeconstructDNApurification
MACHEREY-NAGEL – 03 / 2014, Rev. 0214
4.8 Difficult-to-lyse strains
ForlargeconstructpurificationofGram-positivebacteriaorstrainswithamoreresistantcellwall,itmightbeadvantageoustostartthepreparationwithalysozymetreatment.ProceedtoresuspendthecellpelletinBufferRES-BACcontaining2 mg/mLlysozymeand incubate at 37 °C for 30 minutes.Then,followthelysisprocedureaccordingtotheNucleoBond® Xtra BAC standard protocol.
4.9 Setup of NucleoBond® Xtra BAC Columns
Ideally,theNucleoBond® Xtra Columns are placed into a NucleoBond® Xtra Combi Rack(seeorderinginformation).TheyareheldeitherbythecollarringofthecartridgesorbythePlasticWashers(includedinthekit)toindividuallyadjusttheheightofeachcolumn(seeFigure3).ThePlasticWasherscanalsobeusedtoholdthecolumnsontopof suitablecollection tubesor flasks.TheNucleoBond® Xtra Combi Rack can be used in combination with NucleoBond® PC 100, 500,and2000 as well. Note that the NucleoBond® Xtra Midi Columns can also be placed in the NucleoBond® Rack Large(REF740563).
A B
Figure 3: Setup of NucleoBond® Xtra Columns with the NucleoBond® Xtra Combi Rack A:Setupforclarification,loading,andfirstwashingstep;B:Setupforelution.
LargeconstructDNApurification
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Large construct DNA purification
MACHEREY-NAGEL – 03 / 2014, Rev. 02
4.10 Filtration and loading of the lysate
Afterthealkalinelysis,thesamplehastobeclearedfromcelldebrisandprecipitatetoensurehighplasmidpurityandafastcolumnflowrate.Thisisachievedbypassingthesolution through a NucleoBond® Xtra BAC Column Filter which is provided already inserted into the NucleoBond® Xtra BAC Column.
NucleoBond® Xtra BAC
NucleoBond® Xtra BACColumn Filter
NucleoBond® Xtra BACColumn
TheNucleoBond® Xtra BAC Column Filters are designed to eliminate the cen-trifugationstepafter thealkaline lysis.Theyarepre-wetduringcolumnequilibrationand allow a time-saving simultaneous clearing of bacterial lysate and loading of the NucleoBond® Xtra BAC Column.
Comparedtolysateclearingbycentrifugationorsyringefilters,theNucleoBond® Xtra BAC Column Filter avoidsshearingoflargeDNAconstructssuchasPACsorBACsbythegentledepthfiltereffect(filtrationoccursonthesurfaceofthefilteraswellasinsidethefiltermatrix). Itsspecialmaterialanddesign leadtoaveryrapidpassageof thelysatethroughthefilterandevenverylargelysatevolumescanbeappliedwithouttheriskofclogging.Thisisespeciallyimportantforlow-copyplasmidpurification.However,ifmorethantherecommendedcellmass(seesection4.5,Table2)waslysed,itmightbe advantageous to remove the precipitate by centrifugation (10 min, ≥5000 x g)beforeapplyingthelysatetothecolumnfilter.
4.11 Washing of the column
The high salt concentration of the lysate prevents proteins and RNA from bindingto the NucleoBond® Xtra BAC Column (see section 4.2, Figure 2). However,to remove all trace amounts of contaminants and to purge the dead volume of the NucleoBond® Xtra BAC Column Filters, it is important to wash the column and the filterintwosubsequentwashingsteps.
First,applytheEquilibration Buffer EQU-BACtothefunnelrimofthefiltertowashallresiduallysateoutofthefilterandontothecolumn.Donotjustpourthebufferinsidethefilter.Then,pulloutanddiscardthecolumnfilterorremovethefilterbyturningthecolumn upside down. It is essential to wash the NucleoBond® Xtra BAC Column
Large construct DNA purification
MACHEREY-NAGEL – 03 / 2014, Rev. 0216
withoutthefilterforasecondtimewithWash Buffer WASH-BAC.Thisensureshighestyields with best achievable purity.
4.12 Elution of large construct DNA
Elutioniscarriedoutunderhigh-saltconditionsandbyashiftofpHfrom6.3to8.5.Under thesealkaline conditions, thepositive chargeof theanion-exchange resin isneutralizedandlargeconstructDNAisreleased.TofacilitatethedissociationoflargeconstructDNAfromtheresin,ELU-BAC should be heated to 65–70 °C.Theelutionefficiencycanbeincreasedfurtherbypreventingtheelutionbufferfromcoolingdowntoofast.Therefore,either incubate the column at 50–60 °C during elution or apply the elution buffer in smaller 1–2 mL portions (heated to 65–70 °C).
4.13 Concentration of large construct DNA
ForanydownstreamapplicationitisnecessarytoprecipitatetheDNAandtoremovesaltandalltracesofalcoholsincetheydisturborinhibitenzymaticactivityneededforrestriction or sequencing reactions.
All NucleoBond® Xtra BAC eluates already contain enough salt for an isopropanol precipitation of DNA.As a result, the precipitation is started by directly adding 0.4volumes of isopropanol. To prevent co-precipitation of salt, use room-temperature (18–25 °C) isopropanolonlyanddonotletthelargeconstructDNAsolutiondropintoavialwith isopropanol. Instead, add the isopropanol to the final eluate and mix immediately.
DNAisthencollectedbycentrifugationandwashedwith70%ethanolaccordingtothestandard protocol. Attention should be paid to the drying step. It is very important that all liquid is pipetted off and allowed to evaporate completely at room temperature to reducecontaminationoflargeconstructDNAwithethanol.However,anyover-drying willresultintheDNAbeinghardertodissolveandshould be avoided.
Todissolvelargeconstructscompletely,incubateinanappropriatevolumeofbufferat 4 °C overnight.Useonlywideborepipettetips(withalargeopening),orcut the tip to increase the opening to prevent large constructs from shearing.
ConcentrationofBAC,PAC,orP1constructswithNucleoBond® Finalizer or Finalizer Largeisnotrecommended.TherecoverydropswithincreasingsizeoftheconstructduetotighterbindingoflargeDNAtotheNucleoBond® Finalizer membrane (Use of NucleoBond®Finalizerisonlyrecommendedforvectorsizessmallerthan50kbp)!
4.14 Determination of DNA yield and quality
TheyieldofalargeconstructDNApreparationshouldbeestimatedpriortoandafterthe isopropanol precipitation in order to calculate the recovery after precipitation and tofindthebestvolumetodissolvethepelletin.SimplyuseeitherNucleoBond® Xtra
17
Large construct DNA purification
MACHEREY-NAGEL – 03 / 2014, Rev. 02
BAC Elution Buffer ELU-BAC or the respective low-salt buffer as a blank in yourphotometric measurement.
The nucleic acid concentration of the sample can be calculated from its UVabsorbanceat260nmwhereanabsorbanceof1(1cmpathlength)isequivalentto50μgDNA / mL.Notethattheabsolutemeasuredabsorbanceshouldliebetween0.1and0.7inordertobeinthelinearpartofLambert-Beer´slaw.Diluteyoursampleintherespective buffer if necessary.
The plasmid purity can also be checked by UV spectroscopy.A ratio ofA260/A280 between1.80–1.90andanA260/A230around2.0indicatespureplasmidDNA.AnA260/A280ratioabove2.0isasignfortoomuchRNAinyourpreparation,andanA260/A280 ratiobelow1.8indicatesproteincontamination.
Note that agarosegels can resolveDNAof different sizesonly to anupper limit of20–30kbp.Aaresult,standardagarosegelsarenotsuitabletoassessthequalityofBACDNA.LargeconstructsandgenomicDNAfragmentsrunmoreorlessatthesamespeed and will appear in one broad band.
Sometimesadditional,slowerrunningbandscanbeobservedorDNAisstuckinthegelslotanddoesnotrunatall.ThesearemostlyconformationalisomersorDNAthatwasdenaturedduringthepurification.ThisDNAisusuallylargeconstructDNA,orthesamemixoflargeconstructandgenomicDNAasthemainband,anddoesnotindicateaveryhighcontaminationwithgenomicDNA.
Large constructs like BAC, can be distinguished from genomic DNA on agarosegelsonlybyPulseFieldGelElectrophoresis.ThissystemhasaconstantlychangingelectricalfieldthatleadstodifferentmigrationspeedsofDNAlargerthan30kbp.
Alternatively,theDNAcanberestricted,forexamplewithBamHI,EcoRI,orHindIII,andanalyzedona0.7%TAEagarosegel.ThelargeconstructDNAwithonlyfewrestrictionsitesshowaladderofdistinctbands,whereasthegenomicDNAmightbevisibleasacontinuous smear in the background.
4.15 Convenient stopping points
Cellpelletscaneasilybestoredforseveralmonthsat-20 °C.
Clearedlysatescanbekeptoniceorat4 °Cforseveraldays.
Foroptimalperformance,thecolumnpurificationshouldnotbeinterrupted.However,thecolumnscanbeleftunattendedforseveralhourssincethecolumnsdonotrundry,causingonlysmalllossesinDNAyield.
The eluate can be stored for several days at 4 °C. Note that the eluate should bewarmeduptoroomtemperaturebeforeprecipitatingtheDNAtoavoidco-precipitationof salt.
MACHEREY-NAGEL – 03 / 2014, Rev. 0218
LargeconstructDNApurification
5 Storage conditions and preparation of working solutions
Storage conditions and preparation of working solutions
Allkitcomponentscanbestoredatroomtemperature(18–25°C)andarestableforatleast two years.
Before you start any NucleoBond® Xtra BACpurificationpreparethefollowing:
• DissolvethelyophilizedRNaseA*ofonevialbytheadditionof1mLofBufferRES-BAC.Wearing gloves is recommended. Pipette up and down until theRNaseA isdissolvedcompletely.Transfer theRNaseAsolutionback to thebottle containing Buffer RES-BAC and shake well. Note the date of RNase A addition.ThefinalconcentrationofRNaseA is100μg / mLBufferRES-BAC.StoreBufferRES-BACwithRNaseAat4 °C.Thesolutionwillbestableatthistemperatureforatleast6months.
• BufferLYS-BACshouldbestoredat room temperature (18–25°C)since thecontainingSDSmightprecipitateattemperaturesbelow20°C.Ifprecipitationisobserved,incubatethebottleforseveralminutesatabout30–40°Candmixwell until the precipitate is completely redissolved.
* REF740436.25contains2x100mgofRNaseA.MakesuretodissolveRNaseAofbothvials,eachin1mLofBufferRES-BAC,andtransferthesolutionbackintothebottlecontainingBufferRES-BAC.
19MACHEREY-NAGEL – 03 / 2014, Rev. 02
LargeconstructDNApurification
6 Safety instructionsThe following components of theNucleoBond® Xtra BAC kits contain hazardouscontents. Wear gloves and goggles and follow the safety instructions given in this section.
GHS classification
OnlyharmfulfeaturesneednotbelabeledwithHandPphrasesupto125mLor125g.Mindergefährliche Eigenschaften müssen bis 125 mL oder 125 g nicht mit H- und P-Sätzen gekennzeichnet werden.
Component Hazard contents GHS symbol Hazard phrases
Precaution phrases
Inhalt Gefahrstoff GHS Symbol H-Sätze P-Sätze
LYS-BAC Sodiumhydroxide<2%
Warning 290,315,319
234,280,302+352,305+351+338,332+313,337+313,390,406
Natriumhydroxid < 2 % Achtung
EQU-BAC Buffer salts + ethanol 5–20%
Warning 226 210,233,403+235
Puffersalze + Ethanol 5–20% Achtung
WASH-BAC Buffer salts + ethanol 5–20%
Warning 226 210,233,403+235
Puffersalze + Ethanol 5–20% Achtung
ELU-BAC Buffer salts + isopropanol 10–15%
Warning 226,319 210,233,280,305+351+338,337+313,403+235
Puffersalze + Isopropanol 10–15%
Achtung
RNase A RNaseA,lyophilized
Danger 317,334 261,280,302+352,304+341,333+313,342+311,363
RNase A, lyophilisiert Gefahr
Hazard phrasesH226 Flammable liquid and vapour.
Flüssigkeit und Dampf entzündbar.H 290 May be corrosive to metals.
Kann gegenüber 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 Maycauseallergyorasthmasymptomsorbreathingdifficultiesifinhaled.
MACHEREY-NAGEL – 03 / 2014, Rev. 0220
LargeconstructDNApurification
Kann bei Einatmen Allergie, asthmaartige Symptome oder Atembeschwerden verursachen.
Precaution phrasesP210 Keepawayfromheat,hotsurfaces,sparks,openflamesandotherignition
sources. No smoking.Von Hitze, heißen Oberflächen, Funken, offenen Flammen sowie anderen Zündquellenarten fernhalten. Nicht rauchen.
P233 Keep container tightly closed.Behälter dicht verschlossen halten.
P234 Keep only in original container.Nur im Originalbehälter aufbewahren.
P261 Avoid breathing dust.Einatmen von Staub vermeiden.
P280 Wearprotectivegloves/eyeprotection.Schutzhandschuhe / Augenschutz tragen.
P302+352 IFONSKIN:Washwithplentyofwater/…BEI KONTAKT MIT DER HAUT: Mit viel Wasser/… waschen.
P304+341 IFINHALED:Ifbreathingisdifficult,removetofreshairandkeepatrestinaposition comfortable for breathing.Bei Einatmen: Bei Atembeschwerden an die frische Luft bringen und in einer Position ruhigstellen, die das Atmen erleichtert.
P305+351+338 IFINEYES:Rinsecontinuouslywithwaterforseveralminutes.Removecon-tact lenses if present and easy to do – continue rinsing.BEI KONTAKT MIT DEN AUGEN: Einige Minuten lang behutsam mit Wasser spülen. Vorhandene Kontaktlinsen nach Möglichkeit entfernen. Weiter spülen.
P332+313 IFskinirritationoccurs:Getmedicaladvice/attention.Bei Hautreizung: Ärztlichen Rat einholen / ärztliche Hilfe hinzuziehen.
P333+313 Ifskinirritationoccurs:Getmedicaladvice/attention.Bei Hautreizung: Ärztlichen Rat einholen / ärztliche Hilfe hinzuziehen.
P337+313 Getmedicaladvice/attentionBei anhaltender Augenreizung: Ärztlichen Rat einholen / ärztliche Hilfe hinzuziehen.
P342+311 Ifexperiencingrespiratorysymptoms:CallaPOISONCENTERordoctor/phy-sician.Bei Symptomen der Atemwege: GIFTINFORMATIONSZENTRUM oder Arzt anrufen.
P363 Wash contaminated clothing before reuseKontaminierte Kleidung vor erneutem Tragen waschen.
P390 Absorb spillage to prevent material damage.Verschüttete Mengen aufnehmen, um Materialschäden zu vermeiden.
P403+235 Store in a well ventilated place. Keep cool.Kühl an einem gut belüfteten Ort aufbewahren.
P406 Storeinacorrosiveresistant/…containerwitharesistantinnerliner.In korrosionsbeständigem / (...) Behälter mit korrosionsbeständiger AUskleidung aufbe-wahren.
ForfurtherinformationpleaseseeMaterialSafetyDataSheets (www.mn-net.com). Weiterführende Informationen finden Sie in den Sicherheitsdatenblättern (www.mn-net.com).
21MACHEREY-NAGEL – 03 / 2014, Rev. 02
NucleoBond® Xtra BAC
7 NucleoBond® Xtra BAC purification
1 Prepare a starter culture
Inoculatea3–5mLstartercultureofLBmediumwithasinglecolonypickedfroma freshly streaked agar plate. Make sure that plate and liquid culture contain the appropriate selective antibiotic to guarantee large construct propagation (see section 4.3formoreinformation).Shakeat32–37°Cand~300rpmfor~8h.
2 Prepare a large overnight culture
Inoculatetwo250mLovernightculturesin1000mLErlenmeyerflasksbydilutingthestarterculture1/1000intothegivenvolumesofLBmediumalsocontainingthe appropriate selective antibiotic. Refer to section 4.5 for larger culture volumes if the cultures are known to grow poorly.
Growtheculturesovernightat32–37°Cand200–250rpmfor12–16h.
Note: To utilize the entire large binding capacity of the NucleoBond® Xtra BAC Columns, it is important to provide enough large construct DNA. If you are not sure about the large construct copy number and growth behavior of your host strain, increase the culture volume and decide in step 3 how much cells to use for the preparation. The culture volume recommended below is calculated for a final OD600 of around 3 and should yield around 100 μg of large construct DNA (see to section 4.5 for more information).
2 x 250 mL
3 Harvest bacterial cells
MeasurethecellcultureOD600 and determine the recommended culture volume.
V [mL] = 1500 / OD600
Pellet the cells by centrifugation at4,500–6,000 x g for ≥ 10 min at 4 °C and discard the supernatant completely.
Note: It is of course possible to use more than the recommended amount of cells. In this case increase RES-BAC, LYS-BAC and NEU-BAC buffer volumes proportionally in steps 4, 5, and 7 (see section 4.7 for more information). Additional lysis buffer might have to be ordered separately (see ordering information for NucleoBond® Xtra BAC Buffer Set I, section 8.2). It might be necessary to use a centrifuge for the lysate clarification in step 8 rather than the NucleoBond® Xtra BAC Column Filters.
MACHEREY-NAGEL – 03 / 2014, Rev. 0222
4 Resuspension (Buffer RES-BAC)
Resuspend the cell pellet completely in Resuspension Buffer RES-BAC + RNase A by pipetting up and down or vortexing the cells.
Foranefficientcelllysisitisimportantthatnoclumpsremaininthesuspension.
Note: Increase RES-BAC buffer volume proportionally if more than the recommended cell mass is used (see section 4.7 for information on optimal cell lysis and section 4.8 regarding difficult-to-lyse strains).
60 mL
5 Cell lysis (Buffer LYS-BAC)
Check Lysis Buffer LYS-BAC for precipitated SDS prior to use. If a white precipitate is visible, warm the buffer for several minutes at 30–40°C untilprecipitate is completely dissolved.Then, proceed to cool the buffer down toroomtemperature(18–25°C).
Add Lysis Buffer LYS-BAC to the suspension.
Mix gently by inverting the tube 5 times. Do not vortex as this will shear and release contaminating chromosomal DNA from cellular debris into thesuspension.
Incubatethemixtureatroomtemperature(18–25°C)for5 min.
Warning: Prolonged exposure to alkaline conditions can irreversibly denatureanddegradelargeconstructDNAandliberatecontaminatingchromosomalDNAinto the lysate.
Note: Increase LYS-BAC buffer volume proportionally if more than the recommended cell mass is used (see section 4.7 for information on optimal cell lysis).
60 mL
6 Equilibration (Buffer EQU-BAC)
Equilibrate a NucleoBond® Xtra BAC Column together with the inserted column filter with Equilibration Buffer EQU-BAC.
Apply thebufferonto the rimof thecolumnfilterasshown in the picture and make sure to wet the entire filter.
Allowthecolumntoemptybygravityflow.Thecolumndoes not run dry.
NucleoBond® Xtra BAC
23MACHEREY-NAGEL – 03 / 2014, Rev. 02
30 mL
7 Neutralization (Buffer NEU-BAC)
Add Neutralization Buffer NEU-BAC to the suspension and immediately mix the lysate gently by inverting the tube until the blue sample turns colorless. Do not vortex. Theflaskortubeusedforthisstepshouldnotbefilledmorethantwothirdstoallowhomogeneousmixing.MakesuretoneutralizecompletelytoprecipitatealltheproteinandchromosomalDNA.Thelysateshouldturnfromaslimy,viscousconsistencytoalowviscosity,homogeneoussuspensionofoff-whiteflocculate.Inaddition,theLyseControlshouldturncompletelycolorlesswithoutanytracesof blue.
Incubate lysate on ice for at least 5 min.
Note: Increase NEU-BAC buffer volume proportionally if more than the recommended cell mass is used (see section 4.7 for information on optimal cell lysis).
60 mL
8 Clarification and loading
Make sure to have a homogeneous suspension of the precipitate by inverting the tube 3 times before applying the lysate to the equilibrated NucleoBond® Xtra BACColumnFilter,thusavoidingcloggingofthefilter.
Note: If more than twice the recommended cell mass is used, the capacity of the column filter might be too small to hold all the precipitate. In this case, centrifuge the lysate at ≥ 5,000 x g for at least 10 min and load the supernatant to the equilibrated column filter.
Thelysateissimultaneouslyclearedandloadedontothecolumn.Refillthefilterifmorelysatehastobeloadedthanthefilterisabletohold.Allowthecolumntoemptybygravityflow.
Optional: Final yield might be increased by reloading the lysate flow-through a second time, especially if the amount of DNA is close to the binding capacity of the NucleoBond® Xtra BAC Column.
NucleoBond® Xtra BAC
MACHEREY-NAGEL – 03 / 2014, Rev. 0224
9 Wash column filter and column (Buffer EQU-BAC)
Wash the NucleoBond® Xtra BAC Column Filter and NucleoBond® Xtra BAC Column with Equilibration Buffer EQU-BAC.
Applythebuffer to thefunnelshapedrimof thefilterand make sure it is washing out the lysate remaining inthefilter.Omittingthissteporjustpouringthebufferdirectly inside the funnel may reduce plasmid yield.
15 mL
10 Discard column filter
Either pull out the NucleoBond® Xtra BAC Column Filter or discard it by turning the column upside down.
11 Wash column (Buffer WASH-BAC)
Wash the NucleoBond® Xtra BAC Column with Wash Buffer WASH-BAC. It is important to remove the column filter beforeapplying Buffer Wash-BAC to avoid low purity.
45 mL
12 Elution (Buffer ELU-BAC)
Heat Elution Buffer ELU-BACto65–70°C.
Removethewastecontainerandplacea15mLor50mLcentrifugetube(notprovided) under the column. Elute the large construct DNA with hotElution Buffer ELU-BAC.
Note: The elution efficiency can be increased by preventing the elution buffer from cooling down to fast. Therefore, either incubate the column at 50–60 °C during elution, or apply the elution buffer in smaller 2–3 mL portions (heated to 65–70 °C).
The overall yield can be increased even further by adding a second elution step with an additional 10 mL of hot elution buffer.
Determine large construct yield by UV spectrophotometry before precipitating the DNA in order to adjust desired concentration of DNA in step 15 and calculate the recovery after precipitation.
NucleoBond® Xtra BAC
25MACHEREY-NAGEL – 03 / 2014, Rev. 02
15 mL
13 Precipitation
Add 0.4 volumes of room-temperature isopropanol to precipitate the eluted largeconstructDNA.Vortexwellandletthemixturesitfor2 minutes.
Centrifuge at ≥ 5,000 x g for ≥ 15 min at ≤ room temperature,preferablyat15,000 x g for 30 min at 4 °C. Carefully discard the supernatant.
6 mL
14 Wash and dry DNA pellet
Add room-temperature 70 % ethanol to the pellet and centrifuge at ≥ 5,000 x g,preferably ≥ 15,000 x g for 5 min at room temperature(18–25°C).
5 mL
Carefully remove ethanol completely from the tube with a pipette tip. Allow the pellet to dry at room temperature(18–25°C).
Note: DNA might be harder to dissolve when over-dried.
10–15 min
15 Reconstitute DNA
Add an appropriate volume of buffer TE or sterile H2O to dissolve the pellet. Lessthan500μLcanbeappliediftheyieldisexpectedtobeverylow.
500–1000 μL
Ideally,incubateovernightat4°CtodissolveBACDNAcompletely.
AvoidpipettingupanddownsincelargeDNAconstructsarepronetoshearing.Instead,shakethetubegentlyanduseonlywideborepipettetips(withalargeopening),orcutthetiptoincreasetheopening.
4 °C over night
IfthedissolvedDNApelletisveryviscous,addmorebufferTEorH2Otoensurecompletedissolvingandacorrectquantification.
DeterminelargeconstructyieldbyUVspectrophotometry.
NucleoBond® Xtra BAC
MACHEREY-NAGEL – 03 / 2014, Rev. 0226
8 Appendix
8.1 Troubleshooting
Ifyouexperienceproblemswithreducedyieldorpurity, it isrecommendedtocheckwhichpurificationstepoftheprocedureiscausingtheproblem.
First, the bacterial culture has to be checked for sufficient growth (OD600) in thepresenceofanappropriateselectiveantibiotic(Table1,section 4.3).Second,aliquotsoftheclearedlysate,theflow-through,thecombinedwashingsteps(BufferEQU-BACandBufferWASH-BAC),andtheeluateshouldbekeptforfurtheranalysisbyagarosegel electrophoresis.
Chooseat least1000μLoftheclearedlysate,flow-through,andcombinedwashingstepsaswellas200μLoftheeluate.
Precipitate the nucleic acids by adding 0.7 volumes of isopropanol. Centrifuge thesamples and wash the pellets using 70 % ethanol. Centrifuge again and remove supernatant.Then,airdryfor10minutesanddissolvetheDNAin30μLTEbuffer,pH8.0,andrun20–30μLona1%agarosegel.
Thegelpicturebelow(Figure4)willhelpyoutoaddressthespecificquestionsoutlinedinthefollowingsectionmorequicklyandefficiently.
This shows, for example, the dominant large construct bands which should onlybepresent in theeluateand in the lysatebefore loading,proving that there is largeconstructproduction in your cell culture (lane1). LargeconstructDNA found in thewashfractions,however,narrowsdowntheproblemtowrongorbadwashbuffers(e.g.,wrongpH,buffercomponentsprecipitated,evaporationofliquidduetowrongstorage).
RNA may be visible as a broad band at the bottom of the gel for the lysate and the lysateflow-throughsamples(lanes1and2).Itmayalsooccurinthewashfractionbutshould be absent in the eluate.
Refer to section 4.14(DeterminationofDNAyieldandquality)formoreinformationonadditional options.
LargeconstructDNApurification
27MACHEREY-NAGEL – 03 / 2014, Rev. 02
M 1 2 3 4 5 M: Marker λ HindIII1: Cleared lysate, large construct and
degraded RNA2: Lysateflow-through,nolargeconstruct
DNA,butdegradedRNA3: Washflow-through,nolargeconstruct
DNAorresidualRNA4: Eluate,purelargeconstructDNA5: EcoRI restriction
Figure 4: Exemplary analytical check of NucleoBond® Xtra BAC purification samples Largeconstruct:240kbpBAC,bacterialstrain:E. coliDH5α.20μLofeachprecipitated
samplehasbeenanalyzedona1%agarosegel.EqualamountsoflargeconstructDNAbefore(lane1)andafter(lane4)purificationusingNucleoBond® Xtra BAC are shown with a recovery of > 90 %
LargeconstructDNApurification
MACHEREY-NAGEL – 03 / 2014, Rev. 0228
Problem Possible cause and suggestions
No or low large construct DNAyield
Large construct did not propagate• Check large construct content in the cleared lysate (see
Figure 4).Usecolonies fromfreshplates for inoculationandadd selective antibiotic to plates and media.
Alkaline lysis was insufficient• Too much cell mass was used. Refer to section 4.5–4.7
regarding recommended culture volumes and lysis buffer volumes. Check large construct content in the cleared lysate (see Figure 4).
• Check Buffer LYS-BAC for SDS precipitation before use,especially after storage below 20°C. If necessary, incubatethebottle forseveralminutesat30–40°CandmixwelluntilSDSisredissolved.
SDS- or other precipitates are present in the sample• LoadthecrudelysateontotheNucleoBond® Xtra BAC Column
Filter inserted into the NucleoBond®XtraBACColumn.Thisensures complete removal of SDS precipitates. Incubationof cleared lysates for longer periods of time might lead to formation of a new precipitate. If precipitate is visible, it isrecommended to filter or centrifuge the lysate again beforeloading it onto the NucleoBond® Xtra BAC Column.
Sample / lysate is too viscous• Too much cell mass was used. Refer to section 4.5–4.7
regarding recommended culture volumes and lysis buffer volumes.
• Make sure to mix well after neutralization until the solutionturns colorless, in order to completely precipitate SDS andchromosomal DNA. Otherwise, filtration efficiency and flowrate go down and SDS prevents DNA from binding to thecolumn.
pH or salt concentrations of buffers are too high• Check large construct content in the wash fractions (see
Figure 4).Keepallbufferstightlyclosed.CheckandadjustpHofBufferEQU-BAC(pH6.3)andELU-BAC(pH8.5)withHClorNaOHifnecessary.
LargeconstructDNApurification
29MACHEREY-NAGEL – 03 / 2014, Rev. 02
Problem Possible cause and suggestions
NucleoBond® Xtra BAC Column Filter clogs during filtration
Culture volumes are too large• Refer to section 4.5–4.7 regarding recommended culture
volumes and larger lysis buffer volumes.
Precipitate was not resuspended before loading• Invert crude lysate at least 3 times directly before loading.
Incomplete precipitation step• Make sure to mix well after neutralization to completely
precipitateSDSandchromosomalDNA.
NucleoBond® Xtra BAC Column is blocked or very slow
Sample is too viscous• Do NOT attempt to purify lysate prepared from a culture
volume larger than recommended with standard lysis buffer volumes. Incomplete lysis does not only block the column butcanalsosignificantly reduceyields.Refer tosection 4.5 for recommended culture volumes and section 4.7 for larger culture volumes and adjusted lysis buffer volumes.
• Make sure to mix well after neutralization to completelyprecipitateSDSandchromosomalDNA.
Lysate was not completely cleared• Use the NucleoBond® Xtra BAC Column Filter or centrifuge at
a higher speed or for a longer period of time.
• Precipitates occur during storage.Clear lysate again beforeloading the column.
RNA con-tamination of large con-structDNA
RNase digestion was insufficient• RNase was not added to Buffer RES-BAC or not stored
properly. Add new RNase to Buffer RES-BAC (see section8.2 fororderinginformation).
pH or salt concentration of wash buffer is too low• Check RNA content in the wash fractions (see Figure 4).
Keep all buffers tightly closed. Check pH of Buffer EQU-BAC (pH 6.3) and WASH-BAC (pH 6.3) and adjust with HCl orNaOHifnecessary.
LargeconstructDNApurification
MACHEREY-NAGEL – 03 / 2014, Rev. 0230
Problem Possible cause and suggestions
Genomic DNAcon-tamination of large con-structDNA
DNA visible in the pocket of an agarose gel was mistaken for genomic DNA• Genomic DNA contamination is usually too low to be seen
on an agarose gel. DNA in the gel loading pockets usuallyis denatured large construct DNA. Do not grow the cultureto stationary phase. Allow for longer re-hybridization afterneutralization(i.e.,increaseincubationafteradditionofBufferNEU-BAC)andusemoreTEbufferorH2Otodissolve largeconstruct completely.
Lysis treatment was too harsh• MakesurenottolyseinBufferLYS-BACformorethan5min.
Lysate was mixed too vigorously or vortexed after lysis • Inverttubeforonly5times.DonotvortexafteradditionofLYS-
BAC.
• Use larger tubes or reduce culture volumes for easier mixing.
Lowpurity(A260/A280 <1.8or>2.0)
NucleoBond® Xtra BAC Column Filter was not removed before second washing step • Proteincontenttoohighduetoinaccuratewashing.Remove
the NucleoBond® Xtra BAC Column Filter before performing the second washing step with Buffer WASH-BAC.
Buffer WASH was used instead of Buffer EQU-BAC for the first wash• Buffer EQU-BAC has to be used to wash out the NucleoBond®
XtraColumnFiltertoavoidSDScarry-over.
Only minimal amounts of DNA were loaded onto the column• Excessive free binding capacity requires more extensive
washing. Add an additional washing step with Buffer WASH-BAC.
• Reducelysistime<5min.
LargeconstructDNAPurification
31MACHEREY-NAGEL – 03 / 2014, Rev. 02
Problem Possible cause and suggestions
No nucleic acid pellet formed after precipitation
Pellet was lost• Handletheprecipitatewithcare.Decantsolutionscarefullyor
evenbetter,pipetteoffthesupernatant.DetermineDNAyieldinBufferELU-BAC inorder tocalculate theamountof largeconstructDNAthatshouldberecoveredafterprecipitation.
Large construct DNA might be smeared over the wall of the tube• Dissolve DNA with an appropriate volume of reconstitution
bufferbyrollingthetubeforatleast30min,ideallyforseveralhours.
Nucleic acid did not precipitate• Check type and volumes of precipitating solvent. Make sure
to use at least 0.4 volumes of isopropanol and mix thoroughly.
• Centrifuge for longer periods of time at higher speed.
Nucleic acid pellet is opaque or white instead of clear and glassy
Co-precipitation of salt• Check isopropanol purity and perform precipitation at room
temperature(18–25°C)butmakingsuretocentrifugeat4°C.Donot let the eluate drip from the column into isopropanol,insteadaddisopropanoltothefinaleluateandmiximmediately.
• DissolvethepelletinwaterorTEbuffer.PrecipitateDNAagainbyadding1/10volumeof3MsodiumacetatepH5.0and0.4volumesofisopropanol.Proceedwiththeprecipitationprotocolin this manual.
Nucleic acid pellet does not resuspend in buffer
Pellet was over-dried• Try todissolveathigher temperatures fora longerperiodof
time(e.g.,2hat37°CorovernightatRT),preferablyunderconstantspinning(3D-shaker).
Co-precipitation of salt or residual alcohol• Washthepelletagainwith70%ethanol,orincreasetherecon-
stitution buffer volume.
LargeconstructDNAPurification
MACHEREY-NAGEL – 03 / 2014, Rev. 0232
Problem Possible cause and suggestions
Purifiedlargeconstruct does not perform well in subsequent reactions
Large construct is contaminated with chromosomal DNA or RNA• Refer to the detailed troubleshooting above.
Large construct is contaminated with residual alcohol• Large construct pellet was not dried completely before
dissolving.PrecipitateDNAagain by adding 1/10 volume of3 M sodium acetate pH 5.0 and 0.4 volumes of isopropanol. ProceedwiththeprecipitationprotocolinthismanualanddryDNApelletcompletely.
DNA is degraded• Use only wide bore pipette tips (with a large opening) for
dissolvinglargeconstructstoavoidshearingoftheDNA.Forexample,cutoff1cmofthepipettetiptoincreasetheopening.
• Makesurethatallyourequipment(pipettes,centrifugetubesetc.)iscleanandnuclease-free.
• Donot lyse the samplewithBuffer LYS-BAC formore than5 min.
DNA is not completely re-hybridized • Increase incubation after neutralization (addition of Buffer
NEU-BAC).
DNA is not completely dissolved• UsemoreTEbufferorH2O todissolve largeconstructDNA
pellets after precipitation.
LargeconstructDNApurification
33MACHEREY-NAGEL – 03 / 2014, Rev. 02
8.2 Ordering information
Product REF Pack of
NucleoBond® Xtra BAC 740436 740436
.10
.2510 preps 25 preps
NucleoBond® Xtra Combi Rack 740415 1
NucleoBond® Xtra BAC Buffer Set(BufferRES-BAC,LYS-BAC,NEU-BAC,RNaseA)
740437 1
Buffer RES-BAC(withoutRNaseA)
740440 1000mL
BufferLYS-BAC 740441 1000mL
Buffer NEU-BAC 740442 1000mL
Buffer EQU-BAC 740443 1000mL
Buffer WASH-BAC 740444 1000mL
BufferELU-BAC 740445 600mL
RNase A 740505 740505
.50 50 mg 100 mg
Visitwww.mn-net.com for more detailed product information.
LargeconstructDNApurification
MACHEREY-NAGEL – 03 / 2014, Rev. 0234
8.3 Product use restriction / warranty
NucleoBond® Xtra BAC kit components are intended, developed, designed, andsoldFORRESEARCHPURPOSESONLY,except,however,anyotherfunctionoftheproductbeingexpresslydescribedinoriginalMACHEREY-NAGELproductleaflets.
MACHEREY-NAGEL products are intended for GENERAL LABORATORY USEONLY!MACHEREY-NAGELproductsaresuitedforQUALIFIEDPERSONNELONLY!MACHEREY-NAGEL products shall in any event only be used wearing adequatePROTECTIVE CLOTHING. For detailed information please refer to the respectiveMaterial Safety Data Sheet of the product! MACHEREY-NAGEL products shallexclusivelybeused inanADEQUATETESTENVIRONMENT.MACHEREY-NAGELdoes not assume any responsibility for damages due to improper application of our products inother fieldsofapplication.Applicationon thehumanbody isSTRICTLYFORBIDDEN.Therespectiveuserisliableforanyandalldamagesresultingfromsuchapplication.
DNA/RNA/PROTEINpurificationproductsofMACHEREY-NAGELaresuitablefor IN VITRO-USESONLY!
ONLYMACHEREY-NAGELproductsspeciallylabeledasIVDarealsosuitableforIN VITRO-diagnosticuse.Pleasepayattentiontothepackageoftheproduct.IN VITRO-diagnosticproductsareexpresslymarkedasIVDonthepackaging.
IFTHERE ISNO IVDSIGN,THEPRODUCTSHALLNOTBESUITABLEFOR IN VITRO-DIAGNOSTICUSE!
ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANYCLINICALUSE(INCLUDING,BUTNOTLIMITEDTODIAGNOSTIC,THERAPEUTICAND/ORPROGNOSTICUSE).
Noclaimor representations is intended for itsuse to identifyanyspecificorganismor forclinicaluse (included,butnot limited todiagnostic,prognostic, therapeutic,orbloodbanking).Itisratherintheresponsibilityoftheuseror-inanycaseofresaleofthe products - in the responsibility of the reseller to inspect and assure the use of the DNA/RNA/proteinpurificationproductsofMACHEREY-NAGELforawell-definedandspecificapplication.
MACHEREY-NAGELshallonlyberesponsiblefortheproductspecificationsandtheperformancerangeofMNproductsaccordingtothespecificationsofin-housequalitycontrol,productdocumentationandmarketingmaterial.
ThisMACHEREY-NAGELproductisshippedwithdocumentationstatingspecificationsand other technical information. MACHEREY-NAGEL warrants to meet the statedspecifications.MACHEREY-NAGEL´ssoleobligationandthecustomer´ssoleremedyis 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 conditionsofMACHEREY-NAGEL,whichareprintedonthepricelist.Pleasecontactus if you wish to get an extra copy.
ThereisnowarrantyforandMACHEREY-NAGELisnotliablefordamagesordefectsarising in shipping and handling (transport insurance for customers excluded), orout of accident or improper or abnormal useof this product; defects in products or
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components not manufactured byMACHEREY-NAGEL, or damages resulting fromsuchnon-MACHEREY-NAGELcomponentsorproducts.
MACHEREY-NAGEL makes no other warranty of any kind whatsoever, andSPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OFANY KINDORNATUREWHATSOEVER, DIRECTLYOR INDIRECTLY, EXPRESSOR IMPLIED, INCLUDING, WITHOUT LIMITATION, AS TO THE SUITABILITY,REPRODUCTIVITY, DURABILITY, FITNESS FORA PARTICULAR PURPOSE ORUSE,MERCHANTABILITY,CONDITION,ORANYOTHERMATTERWITHRESPECTTOMACHEREY-NAGELPRODUCTS.
In no event shall MACHEREY-NAGEL be liable for claims for any other damages,whether direct, indirect, incidental, compensatory, foreseeable, consequential, orspecial(includingbutnotlimitedtolossofuse,revenueorprofit),whetherbaseduponwarranty,contract,tort(includingnegligence)orstrictliabilityarisinginconnectionwiththesaleorthefailureofMACHEREY-NAGELproductstoperforminaccordancewiththestatedspecifications.ThiswarrantyisexclusiveandMACHEREY-NAGELmakesno other warranty expressed or implied.
The warranty provided herein and the data, specifications and descriptions of thisMACHEREY-NAGELproductappearinginMACHEREY-NAGELpublishedcataloguesand product literature are MACHEREY-NAGEL´s sole representations concerningtheproductandwarranty.Nootherstatementsorrepresentations,writtenororal,byMACHEREY-NAGEL´semployees,agentorrepresentatives,exceptwrittenstatementssignedbyadulyauthorizedofficerofMACHEREY-NAGELareauthorized;theyshouldnot be relied upon by the customer and are not a part of the contract of sale or of this warranty.
Productclaimsaresubjecttochange.ThereforepleasecontactourTechnicalServiceTeam for the most up-to-date information on MACHEREY-NAGEL products. Youmayalsocontactyour localdistributor forgeneralscientific information.Applicationsmentioned inMACHEREY-NAGEL literatureareprovided for informationalpurposesonly.MACHEREY-NAGELdoesnotwarrantthatallapplicationshavebeentestedinMACHEREY-NAGELlaboratoriesusingMACHEREY-NAGELproducts.MACHEREY-NAGELdoesnotwarrantthecorrectnessofanyofthoseapplications.
Lastupdated:07/2010,Rev.03
Pleasecontact: MACHEREY-NAGELGmbH&Co.KG Tel.:+49(0)2421969270 e-mail: [email protected]
LargeconstructDNApurification