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Plasmid DNA Purification

MACHEREY-NAGEL – 01/2008/ Rev. 04

64

Problem

Possible cause and suggestions

RNA contami-
nation of
plasmid DNA

RNase digestion was inefficient

•

 

RNase was not added to Buffer RES or stored improperly. Add
new RNase to Buffer RES. See section 8.2 for ordering informa-
tion.

PH or salt concentration of wash buffer is too low

•

 

Check RNA content in the wash fractions (see Figure 6). Keep
all buffers tightly closed. Check pH of Buffer EQU (pH 6.5) and
WASH (pH 6.5) and adjust with HCl or NaOH if necessary.

Low purity
(A

260

/A

280

< 1.8)

NucleoBond

® 

Xtra column filter was not removed before second

washing step

•

 

Protein content too high due to inefficient washing. Remove the
NucleoBond

® 

Xtra column filter 

before

 performing the second

washing step with Buffer WASH.

Buffer WASH was used instead of Buffer EQU for the first wash

•

 

Buffer EQU has to be used to wash out the NucleoBond

® 

Xtra

column filter to avoid SDS carryover.

Only minimal amounts of DNA were loaded onto the column

•

 

Too much free binding capacity needs more extensive washing
– double washing step with Buffer WASH.

•

 

Reduce lysis time < 5 min.

No nucleic
acid pellet
formed after
precipitation

Pellet was lost

•

 

Handle the precipitate with care. Decant solutions carefully. De-
termine DNA yield in Buffer ELU in order to calculate the plas-
mid DNA that should be recovered after precipitation.

Plasmid DNA might be smeared over the wall of the tube

•

 

Dissolve DNA with an appropriate volume of reconstitution buffer
by rolling the tube for at least 30 min.

Nucleic acid did not precipitate

•

 

Check type and volumes of precipitating solvent. Make sure to
use at least 0.7 volumes of isopropanol and mix thoroughly.

•

 

Centrifuge for longer periods of time at higher speed.

Содержание NucleoBond Xtra Midi

Страница 1: ...id DNA Purification User Manual NucleoBond Xtra Midi NucleoBond Xtra Maxi NucleoBond Xtra Midi Plus NucleoBond Xtra Maxi Plus January 2008 Rev 04 Procedure in English German French NEW MACHEREY NAGEL...

Страница 2: ...eoBond Xtra column filter discard NucleoBond Xtra column filter discard NucleoBond Xtra column filter 8 2nd Washing Buffer WASH 8 ml Buffer WASH 25 ml 9 Elution Buffer ELU 5 ml Buffer ELU 15 ml 10 Pre...

Страница 3: ...9 Filtration and loading of the lysate 17 4 10 Washing of the column 17 4 11 Elution and concentration of plasmid DNA 18 4 12 Determination of DNA yield and quality 21 4 13 Convenient stopping points...

Страница 4: ...purification Midi Maxi French 47 7 8 Low copy plasmid purification Midi Maxi French 53 7 9 Concentration of NucleoBond Xtra eluates with the NucleoBond Finalizers French 57 8 Appendix 60 8 1 Troubles...

Страница 5: ...EU 100 ml 500 ml 1000 ml 100 ml 500 ml Buffer EQU 200 ml 2 x 500 ml 2 x 1000 ml 200 ml 2 x 500 ml Buffer WASH 100 ml 500 ml 1000 ml 100 ml 500 ml Buffer ELU 60 ml 300 ml 600 ml 60 ml 300 ml RNase A ly...

Страница 6: ...150 ml 750 ml Buffer EQU 500 ml 2 x 1000 ml 500 ml 5 x 1000 ml 500 ml 2 x 1000 ml 500 ml Buffer WASH 300 ml 1000 ml 500 ml 3 x 1000 ml 300 ml 1000 ml 500 ml Buffer ELU 180 ml 900 ml 2 x 900 ml 180 ml...

Страница 7: ...ipment for growing and harvesting bacteria e g inoculating loop culture tubes and flasks 37 C shaking incubator and centri fuge with rotor and tubes or bottles for harvesting cells Refrigerated centri...

Страница 8: ...llow a time saving simultaneous clearing of bacterial lysate and loading of cleared lysate onto the Nu cleoBond Xtra column Furthermore the use of the column filters avoids the time consuming centrifu...

Страница 9: ...ysis Buffer LYS to the suspension Mix gently by inverting the tube 5 times Do not vortex as this will shear and release contaminating chromosomal DNA from cellular debris into the suspension Incubate...

Страница 10: ...is a silica based anion exchange resin developed by MACHEREY NAGEL and covered under European Patent EP 0 496 822 It is devel oped for routine separation of different classes of nucleic acids like oli...

Страница 11: ...rom proteins to RNA are washed from the column the positive charge of the resin is neutralized by a pH shift to slightly alkaline conditions and pure plasmid DNA is eluted in a high salt elution buffe...

Страница 12: ...or each host strain plasmid construct combination individually Cell cultures should be grown under antibiotic selection at all times to ensure plas mid propagation Without this selective pressure cell...

Страница 13: ...t weight Overnight cultures grown in LB medium usually reach an OD600 of 3 6 under vigorous shaking in flasks Fermentation cultures even reach an OD600 of 10 and more The expected DNA yield for a high...

Страница 14: ...tures for detailed information on calculating ODV OD600 x Vol refer to section 4 4 For higher yields it is advantageous to increase the cell culture and lysis buffer vol umes even more e g by factor 3...

Страница 15: ...ulture volumes in case of e g low copy vector purification section 4 5 Table 3 By rule of thumb calculate the necessary lysis buffer volumes for RES LYS and NEU as follows Vol ml Culture Volume ml x O...

Страница 16: ...individually adjust the height of each column see Figure 3 The Plastic Washers can also be used to hold the columns on top of suitable collecting tubes or flasks The NucleoBond Xtra Combi Rack can be...

Страница 17: ...er and even very large lysate volumes can be applied without the risk of clogging This is especially important for e g low copy plasmid purification However if more than the recommended cell mass see...

Страница 18: ...alination of plasmid and cosmid DNA eluates that are obtained by anion exchange chromatog raphy based DNA purification The sample is precipitated with isopropanol as men tioned above and loaded onto a...

Страница 19: ...ved by using 600 l of elution buffer For a higher concentration experienced users can lower the elution buffer volume to 400 200 l Table 4 gives an overview about recovery and concentration of differe...

Страница 20: ...l of elution buffer For a higher concentration experienced users can lower the elution buffer volume to 600 400 l Table 5 gives an overview about recovery and concentration of different amounts of pl...

Страница 21: ...ure plasmid DNA An A260 280 ra tio above 2 0 is a sign for too much RNA in your preparation an A260 280 ratio below 1 8 indicates protein contamination Plasmid quality can be checked by running the pr...

Страница 22: ...Nase A solution back to the bottle con taining Buffer RES and shake well Note the date of RNase A addition The fi nal concentration of RNase A is 60 g ml Buffer RES Store Buffer RES with RNase A at 4...

Страница 23: ...Phrases R 36 38 Irritating to eyes and skin R 42 43 May cause sensitization by inhalation and skin contact Safety Phrases S 22 Do not breathe dust S 24 Avoid contact with the skin S 26 In case of cont...

Страница 24: ...C and 300 rpm for 12 16 h Note To utilize the entire large binding capacity of the NucleoBond Xtra columns it is important to provide enough plasmid DNA If you are not sure about the plasmid copy numb...

Страница 25: ...inutes at 30 40 C until precipitate is dis solved completely Cool buffer down to room temperature 20 25 C Add Lysis Buffer LYS to the suspension Mix gently by inverting the tube 5 times Do not vortex...

Страница 26: ...precipitate by inverting the tube 3 times directly before applying the lysate to the equilibrated NucleoBond Xtra column filter to avoid clogging of the filter The lysate is simultaneously cleared an...

Страница 27: ...ra column with Wash Buffer WASH It is important to remove the column filter before applying the washing buffer to avoid low purity 8 ml 25 ml Elution Buffer ELU Elute the plasmid DNA with Elution Buff...

Страница 28: ...g preferably 15 000 x g for 5 min at room temperature 20 25 C 2 ml 5 ml Carefully remove ethanol completely from the tube with a pipette tip Allow the pellet to dry at room temperature 20 25 C Note P...

Страница 29: ...ond Xtra columns it is important to provide enough plasmid DNA For the standard low copy procedure the cul ture volumes were doubled compared to the high copy vector protocol However due to a plasmid...

Страница 30: ...eral minutes at 30 40 C until precipitate is dis solved completely Cool buffer down to room temperature 20 25 C Add Lysis Buffer LYS to the suspension Mix gently by inverting the tube 5 times Do not v...

Страница 31: ...homogeneous mixing Make sure to neutralize completely to precipitate all the protein and chromosomal DNA The lysate should turn from a slimy viscous consistency to a low viscosity homogeneous suspensi...

Страница 32: ...smid content of your eluate prior to the precipitation step by measuring A260 see section 4 12 Load ing more DNA might lead to clogging and complete loss of your sample 3 5 ml for 5 ml eluate 10 5 ml...

Страница 33: ...he DNA 5 Elute DNA Remove the NucleoBond Finalizer from the syringe pull out the plunger of a 1 ml Syringe and attach the NucleoBond Finalizer to the syringe outlet Note Refer to section 4 11 Table 4...

Страница 34: ...es wichtig ausreichend Plasmid DNA zu laden Sollten Sie bzgl Kopienzahl des Plasmids und Wachstumsverhaltens des Bakterienstamms unsicher sein erh hen Sie das Kulturvolumen und entscheiden in Schritt...

Страница 35: ...fer LYS Vor Gebrauch berpr fen Sie Lysis Buffer LYS auf ausgefallenes SDS Sollte ein wei es Pr zipitat sichtbar sein erw rmen Sie den Puffer f r einige Minuten auf 30 40 C bis das Pr zipitat komplett...

Страница 36: ...en Sie sofort aber vorsichtig durch 10 bis 15 maliges Invertieren Vortexen Sie nicht Das f r diesen Schritt verwendete Gef sollte nicht mehr als zwei Drittel gef llt sein um ein gleichm iges Durchmisc...

Страница 37: ...ie die Zentri fugation vorzugsweise bei h herer Geschwindigkeit oder geben Sie ihn auf den NucleoBond Xtra Filter Diese Lysatkl rung ist sehr wichtig da restliches Pr zipitat die NucleoBond Xtra S ule...

Страница 38: ...z B BACs f hren Fahren Sie mit Schritt 13 fort um die Isopropanol F llung gem Zentrifugati onsprotokoll durchzuf hren oder folgen Sie der Anleitung in Kapitel 7 6 zur Kon zentrierung und Entsalzung mi...

Страница 39: ...eratur 20 25 C an der Luft trocknen Hinweis Zu langes Trocknen des Pellets kann das anschlie ende L sen der DNA er schweren 5 10 min 10 15 min 15 L sen der DNA L sen Sie das DNA Pellet in einem geeign...

Страница 40: ...unten angegebenen Volumens durch Verd nnen der Vorkultur um den Faktor 1 1000 Stellen Sie sicher dass das Medium das n tige Antibiotikum enth lt W hlen Sie ein gr eres Volumen Kapitel 4 5 falls die K...

Страница 41: ...na der Puffer RES LYS und NEU proportio nal in den Schritten 4 5 and 7 Eventuell m ssen zus tzliche Volumina der Lysispuffer bestellt werden s Bestellinformation f r das NucleoBond Xtra Buffer Set I K...

Страница 42: ...tzung aus den Zelltr mmern in die Suspension f hren kann Inkubieren Sie die Mischung f r 5 min bei Raumtemperatur 20 25 C Hinweis Erh hen Sie das Volumen des Puffers LYS proportional falls mehr als di...

Страница 43: ...dass die Neutralisation vollst ndig erfolgt ist um eine quantitative F llung von Protein und genomischer DNA zu gew hrleisten Das schleimige viskose Lysat sollte nach Zugabe des Puffers NEU d nnfl ssi...

Страница 44: ...or der Pr zipitation durch Bestimmung des A260 siehe Kapitel 4 12 Die Beladung mit gr eren DNA Mengen kann zum Verstopfen des NucleoBond Finalizers und somit zum kompletten Verlust Ihrer Probe f hren...

Страница 45: ...r f r 10 Minuten bei 80 C Zu intensives Trocknen der DNA kann allerdings zu einer reduzierten Wiederfindung f hren 5 Elution der DNA Entfernen Sie den NucleoBond Finalizer von der 30 ml Spritze ziehen...

Страница 46: ...1 2008 Rev 04 46 Midi NucleoBond Finalizer Maxi NucleoBond Finalizer Large 6 Bestimmung der Plasmid Ausbeute Bestimmen Sie photometrisch die Plasmidausbeute und berpr fen Sie die Plasmidintegrit t mit...

Страница 47: ...n tes pas s r du nombre de copies de votre plasmide ou du comportement de la culture vis vis de la croissance bact rienne augmentez le volume de culture et d cidez l tape 3 du nombre de cellules utili...

Страница 48: ...es lyser 8 ml 12 ml 5 Lyse cellulaire Buffer LYS Avant d utiliser le tampon LYS v rifiez que le SDS n a pas pr cipit Si un pr cipit blanc est visible chauffez le tampon quelques minutes 30 40 C jusqu...

Страница 49: ...imm diatement avec pr caution le lysat en inversant le tube 10 15 fois Ne pas utiliser de vortex Le flacon ou le tube utilis pour cette tape ne doit pas tre rempli au plus des deux tiers afin de perm...

Страница 50: ...gation de pr f rence une vitesse plus lev e ou chargez le lysat sur le filtre NucleoBond Xtra pr alablement quilibr Cette tape de clarification est extr mement importante En effet les r sidus du pr ci...

Страница 51: ...taille comme des BACs Passez l tape 13 pour le protocole de centrifugation apr s la pr cipitation l isopropanol ou continuez avec la section 7 9 pour la concentration de l ADN plasmidique et son d sal...

Страница 52: ...ure ambiante 20 25 C Remarque l ADN plasmidique peut tre plus difficile dissoudre si le culot est tr s sec 5 10 min 10 15 min 15 Reconstitution de l ADN Dissoudre le culot d ADN dans un volume ad quat...

Страница 53: ...B contenant l antibiotique s lectif appropri Si la culture pr sente une faible croissance ou si le plasmide est connu pour tre faiblement repr sent consultez la section 4 5 du protocole pour l utilisa...

Страница 54: ...7 ventuellement il faudra commander un volume suppl mentaire de tampon de lyse voir I information de commande du Nu cleoBond Xtra Buffer Set I chapitre 8 2 Utilisez la centrifugation pour la clarific...

Страница 55: ...tionnerait se d tacherait des d bris cellulaires et contaminerait alors la suspension Incubez le m lange temp rature ambiante 20 25 C pendant 5 min Remarque augmentez proportionnellement le volume de...

Страница 56: ...ttre un m lange homog ne Veillez neutraliser compl tement pour pr cipiter toutes les prot ines et l ADN chromosomique Le lysat doit passer d une forme gluante et visqueuse une forme moins visqueuse un...

Страница 57: ...nu d ADN plasmidique de vos luats avant de passer l tape de pr cipitation en mesurant A260 voir la section 4 12 Charger plus d ADN peut entra ner une surcharge et une perte compl te de l chantillon 3...

Страница 58: ...hanolique Cependant le taux de r cup ration final peut tre r duit en raison d un s chage excessif de l ADN 5 Elution de l ADN Enlevez le NucleoBond Finalizer de la seringue retirez le piston d une ser...

Страница 59: ...Rev 04 59 Midi NucleoBond Finalizer Maxi NucleoBond Finalizer Large 6 D termination du rendement D terminez le rendement d ADN plasmidique par spectrophotom trie UV et confirmez l int grit du plasmid...

Страница 60: ...n a 1 agarose gel Table 6 NucleoBond Xtra eluate volumes required for an analytical check Volume required l Sample Purification step Midi Maxi I Cleared lysate of protocol step 8 500 200 II Column flo...

Страница 61: ...1 agarose gel Equal amounts of plasmid DNA before lane 1 and after lane 4 purification using NucleoBond Xtra Midi are shown with a recovery of 90 M 1 2 3 4 5 M Marker HindIII 1 I cleared lysate ccc li...

Страница 62: ...olved SDS or other precipitates are present in the sample Load the crude lysate onto the NucleoBond Xtra column filter inserted in the NucleoBond Xtra column This ensures com plete removal of SDS prec...

Страница 63: ...olumn size with stan dard lysis buffer volumes Incomplete lysis not only blocks the column but can also significantly reduce yields Refer to section 4 4 and 4 5 for recommended culture volumes and sec...

Страница 64: ...er WASH Buffer WASH was used instead of Buffer EQU for the first wash Buffer EQU has to be used to wash out the NucleoBond Xtra column filter to avoid SDS carryover Only minimal amounts of DNA were lo...

Страница 65: ...o detailed trouble shooting No or low plasmid DNA yield Dead volume too high Especially when you aim for high concentration you need to elute in small volumes But naturally you will lose parts of your...

Страница 66: ...can be ex pected Fresh elution buffer was used for second elution step The second elution step is crucial for optimal yield but to achieve a high DNA concentration the eluate of the first elution step...

Страница 67: ...tinued DNA is degraded Make sure that your entire equipment pipettes centrifuge tubes etc is clean and nuclease free Do not lyse the sample with Buffer LYS for more than 5 min DNA is irreversibly dena...

Страница 68: ...lus For use with NucleoBond Xtra Maxi Maxi EF 740419 20 20 large filters 20 syringe sets RNase A 740505 100 mg RNase A 740505 50 50 mg 8 3 Product use restriction warranty NucleoBond Xtra Midi Maxi ki...

Страница 69: ...in connection with the sale or the failure of MA CHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty ex...

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