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NucleoBond® Xtra BAC - MACHEREY

1. Large construct DNA purification User manual NucleoBond Xtra BAC March 2014 Rev 02 MACHEREY NAGEL www mn net com SUN Large construct DNA purification NucleoBond Xtra BAC Protocol at a glance Rev 02 1 Prepare a starter culture 2 Prepare a large overnight culture 3 Harvest bacterial cells 4 500 6 000 x g gt 10 min at 4 C 4 Resuspension 60 mL Buffer RES BAC 5 Cell Iysis Important Check Buffer LYS BAC for precipitated SDS 6 Equilibration of the column and filter 60 mL Buffer LYS BAC Invert the tube 5 times RT 5 min 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 gt 25 min 8 Clarification and loading of the Iysate Invert the tube 3 times Load lysate on NucleoBond Xtra BAC Column Filter 9 Wash column filter and column 15 mL Buffer EQU BAC EU 7 10 Discard column filter Discard NucleoBond Xtra BAC Column Filter 11 Wash column 45 mL Buffer WASH BAC EU 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 H O pH 7 MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8
2. 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 You will find volumes or incubation times in the white boxes The name of the buffer as well as incubation times repeats or important handling steps are emphasized in bold type within the instructions Additional notes or optional steps are printed in italic The exclamation point marks information and hints that are essential for a successful preparation In the example shown above you are asked to check the Lysis Buffer LYS BAC prior to use 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 MACHEREY NAGEL 03 2014 Rev 02 7 Large construct DNA purification 4 NucleoBond Xtra BAC plasmid purification system 4 1 Basic principle The bacterial cells are lysed by an optimized set of newly formulated buffers based on the NaOH SDS lysis method of Birnboim and Doly After equilibration of the NucleoBond Xtra BAC Column together with the corresponding NucleoBond Xtra BAC Column Filter the entire lysate is loaded by gravity flow and simultaneously cleared by the specially designed column filter Plasmid DNA is bound to the NucleoBond Xtra BAC Silica Resin Upon completion of the washing step the plasmid DNA is eluted precipitated and e
3. SDS or other precipitates are present in the sample Load the crude lysate onto the NucleoBond Xtra BAC Column Filter inserted into the NucleoBond Xtra BAC Column This ensures complete removal of SDS precipitates Incubation of cleared lysates for longer periods of time might lead to formation of a new precipitate If precipitate is visible it is recommended to filter or centrifuge the lysate again before loading 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 solution turns colorless in order to completely precipitate SDS and chromosomal DNA Otherwise filtration efficiency and flow rate go down and SDS prevents DNA from binding to the column PH or salt concentrations of buffers are too high Check large construct content in the wash fractions see Figure 4 Keep all buffers tightly closed Check and adjust pH of Buffer EQU BAC pH 6 3 and ELU BAC pH 8 5 with HCl or NaOH if necessary 28 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA purification Problem Possible cause and suggestions Culture volumes are too large Refer to section 4 5 4 7 regarding recommended culture volumes and larger lysis buffer volumes NucleoBond Xtra BAC Precipitate was not resuspended before loading Column Filter f 7
4. ethanol according to the standard 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 reduce contamination of large construct DNA with ethanol However any over drying will result in the DNA being harder to dissolve and should be avoided To dissolve large constructs completely incubate in an appropriate volume of buffer at 4 C overnight Use only wide bore pipette tips with a large opening or cut the tip to increase the opening to prevent large constructs from shearing Concentration of BAC PAC or P1 constructs with NucleoBond Finalizer or Finalizer Large is not recommended The recovery drops with increasing size of the construct due to tighter binding of large DNA to the NucleoBond Finalizer membrane Use of NucleoBond Finalizer is only recommended for vector sizes smaller than 50 kbp 4 14 Determination of DNA yield and quality The yield of a large construct DNA preparation should be estimated prior to and after the isopropanol precipitation in order to calculate the recovery after precipitation and to find the best volume to dissolve the pellet in Simply use either NucleoBond Xtra 16 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA purification BAC Elution Buffer ELU BAC or the respective low salt buffer as a blank in your photometric measurement The nucleic acid concentration of the sample
5. 52355 D ren Germany Tel 49 24 21 969 270 Fax 49 24 21 969 199 tech bio mn net com www mn net com Large construct DNA purification Table of contents 1 Components 1 1 Kit contents 1 2 Reagents and equipment to be supplied by user 2 Kit specifications 6 3 About this user manual 7 4 NucleoBond Xtra BAC plasmid purification system 4 1 Basic principle 4 2 NucleoBond Xtra BAC anion exchange columns 4 3 Growth of bacterial cultures 10 4 4 Chloramphenicol amplification of low copy plasmids 11 4 5 Culture volume for large constructs 12 4 6 Lysate neutralization and LyseControl 12 4 7 Cell lysis 13 4 8 Difficult to Iyse strains 14 4 9 Setup of NucleoBond Xtra BAC Columns 14 4 10 Filtration and loading of the lysate 15 4 11 Washing of the column 15 4 12 Elution of large construct DNA 16 4 13 Concentration of large construct DNA 16 4 14 Determination of DNA yield and quality 16 4 15 Convenient stopping points 17 5 Storage conditions and preparation of working solutions 18 6 Safety instructions 19 7 NucleoBond Xtra BAC purification 21 8 Appendix 26 8 1 Troubleshooting 26 8 2 Ordering information 33 8 3 Product use restriction warranty 34 MACHEREY NAGEL 03 2014 Rev 02 3 Large construct DNA purification 1 Components 1 1 Kit contents NucleoBonga Xtra BAC 10 preps 25 preps REF 740436 10 740436 25 Buffer RES BAC 750 mL 2 x 1000 mL Buffer LYS BAC 750 mL 2 x 1000 mL Buffer NEU BAC 750 mL 2 x
6. clogs during Invert crude lysate at least 3 times directly before loading filtration Incomplete precipitation step Make sure to mix well after neutralization to completely precipitate SDS and chromosomal DNA Sample is too viscous Do NOT attempt to purify lysate prepared from a culture volume larger than recommended with standard Iysis buffer volumes Incomplete lysis does not only block the column but can also significantly reduce yields Refer to section 4 5 NucleoBond for recommended culture volumes and section 4 7 for larger ER culture volumes and adjusted lysis buffer volumes Xtra BAC Column is Make sure to mix well after neutralization to completely blocked or precipitate SDS and chromosomal DNA very slow 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 before loading the column RNase digestion was insufficient RNase was not added to Buffer RES BAC or not stored properly Add new RNase to Buffer RES BAC see section 8 2 RNA con for ordering information tamination of large con PH or salt concentration of wash buffer is too low struct DNA 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 HCI or NaOH if necessary MACHEREY NAGEL 03 2014 Rev 02 29 Large
7. 1000 mL Buffer EQU BAC 500 mL 1000 mL 500 mL Buffer WASH BAC 500 mL 1000 mL 250 mL Buffer ELU BAC 200 mL 500 mL RNase A lyophilized 75 mg 2 x 100 mg NucleoBond Xtra BAC Columns 10 25 incl NucleoBond Xtra BAC Column Filters Plastic Washers 5 10 User manual 1 1 For preparation of working solutions and storage conditions see section 5 4 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA purification 1 2 Reagents and equipment to be supplied by user Reagents e Isopropanol room temperatured 70 ethanol room temperatured Buffer for reconstitution of DNA for example TE buffer or sterile H2O pH 7 Equipment Standard microbiological equipment for growing and harvesting bacteria e g inoculating loop culture tubes and flasks 37 C shaking incubator and centrifuge with rotor and tubes or bottles for harvesting cells Refrigerated centrifuge capable of reaching 5 000 x g with rotor for the appropriate centrifuge tubes or bottles Centrifugation tubes or vessels with suitable capacity for the volumes specified in the respective protocol NucleoBond Xtra Combi Rack see ordering information or equivalent holder MACHEREY NAGEL 03 2014 Rev 02 5 Large construct DNA purification Kit specifications NucleoBond Xtra BAC is designed for ultra fast purification of cosmids and very large constructs P1 BACs PACs ranging from 3 kbp up to 300 kbp For preparation of working solutions
8. 2 Warning 290 315 234 280 Natriumhydroxid lt 2 gt Achtung 319 302 352 305 351 338 332 313 337 313 390 406 EQU BAC Buffer salts ethanol Warning 226 210 233 5 20 403 235 Puffersalze Ethanol 5 20 Achtung WASH BAC Buffer salts ethanol Warning 226 210 233 5 20 403 235 Puffersalze Ethanol 5 20 Achtung ELU BAC Buffer salts isopropanol DD Warning 226 319 210 233 280 10 15 305 351 338 Puffersalze Isopropanol Achtung 337 313 10 15 403 235 RNase A RNase A Iyophilized Danger 317 334 261 280 RNase A Iyophilisiert D Gefahr 302 352 304 341 333 313 342 311 363 Hazard phrases H 226 Flammable liquid and vapour Fl ssigkeit und Dampf entz ndbar H 290 May be corrosive to metals Kann gegen ber Metallen korrosiv sein H 315 Causes skin irritation Verursacht Hautreizungen H 317 May cause an allergic skin reaction Kann allergische Hautreaktionen verursachen H 319 Causes serious eye irritation Verursacht schwere Augenreizung H 334 May cause allergy or asthma symptoms or breathing difficulties if inhaled MACHEREY NAGEL 03 2014 Rev 02 19 Large construct DNA purification Kann bei Einatmen Allergie asthmaartige Symptome oder Atembeschwerden verursachen Precaution phrases P 210 P 233 P 234 P 261 P 280 P 302 352 P 304 341 P 305 351 338 P 332 313 P 333 313 P 337 313 P 342 311 P 363 P 390 P 403 235
9. CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL
10. EQU BAC and Buffer WASH BAC and the eluate should be kept for further analysis by agarose gel electrophoresis Choose at least 1000 uL of the cleared Iysate flow through and combined washing steps as well as 200 uL of the eluate Precipitate the nucleic acids by adding 0 7 volumes of isopropanol Centrifuge the samples and wash the pellets using 70 ethanol Centrifuge again and remove supernatant Then air dry for 10 minutes and dissolve the DNA in 30 uL TE buffer pH 8 0 and run 20 30 uL on a 1 agarose gel The gel picture below Figure 4 will help you to address the specific questions outlined in the following section more quickly and efficiently This shows for example the dominant large construct bands which should only be present in the eluate and in the lysate before loading proving that there is large construct production in your cell culture lane 1 Large construct DNA found in the wash fractions however narrows down the problem to wrong or bad wash buffers e g wrong pH buffer components precipitated evaporation of liquid due to wrong storage RNA may be visible as a broad band at the bottom of the gel for the lysate and the lysate flow through samples lanes 1 and 2 It may also occur in the wash fraction but should be absent in the eluate Refer to section 4 14 Determination of DNA yield and quality for more information on additional options 26 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA p
11. P 406 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking Von Hitze hei en Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen Keep container tightly closed Beh lter dicht verschlossen halten Keep only in original container Nur im Originalbeh lter aufbewahren Avoid breathing dust Einatmen von Staub vermeiden Wear protective gloves eye protection Schutzhandschuhe Augenschutz tragen IF ON SKIN Wash with plenty of water BEI KONTAKT MIT DER HAUT Mit viel Wasser waschen IF INHALED If breathing is difficult remove to fresh air and keep at rest ina position comfortable for breathing Bei Einatmen Bei Atembeschwerden an die frische Luft bringen und in einer Position ruhigstellen die das Atmen erleichtert IF IN EYES Rinse continuously with water for several minutes Remove con tact lenses if present and easy to do continue rinsing BEI KONTAKT MIT DEN AUGEN Einige Minuten lang behutsam mit Wasser sp len Vorhandene Kontaktlinsen nach M glichkeit entfernen Weiter sp len IF skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen If skin irritation occurs Get medical advice attention Bei Hautreizung Arztlichen Rat einholen rztliche Hilfe hinzuziehen Get medical advice attention Bei anhaltender Augenreizung Arztlichen Rat ein
12. and storage conditions see section 5 NucleoBond Xtra BAC Columns are polypropylene columns containing NucleoBond Xtra BAC Silica Resin packed between two inert filter elements The columns offer a binding capacity of 200 ug for very large constructs All NucleoBond Xtra BAC Columns are resistant to organic solvents such as alcohol chloroform and phenol and are also suitable for buffers containing denaturing agents like formamide urea or common detergents like Triton X 100 or NP 40 NucleoBond Xtra BAC Silica Resin can be used over a wide pH range pH 2 5 8 5 and can remain in contact with buffers for several hours without any change in its chromatographic properties The NucleoBond Xtra BAC Column Filters are specially designed depth filters that fit into the NucleoBond Xtra BAC Columns These filters are inserted and ready to use in the NucleoBond Xtra BAC Columns This allows for a time saving simultaneous clearing of bacterial lysate and loading of cleared lysate onto the NucleoBond Xtra BAC Column As a result the time consuming centrifugation step for lysate clearing is avoided The NucleoBond Xtra BAC Column Filters allow complete removal of precipitate without clogging even with large lysate volumes and also avoid shearing of large DNA constructs such as PACs or BACs LyseControl The Lysis Buffer LYS BAC contains a blue pH indicator to ensure complete neutralization for maximum yield MACHEREY NAGE
13. construct DNA Purification Problem Possible cause and suggestions Genomic DNA con tamination of large con struct DNA 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 usually is denatured large construct DNA Do not grow the culture to stationary phase Allow for longer re hybridization after neutralization i e increase incubation after addition of Buffer NEU BAC and use more TE buffer or H O to dissolve large construct completely Lysis treatment was too harsh Make sure not to lyse in Buffer LYS BAC for more than 5 min Lysate was mixed too vigorously or vortexed after lysis Invert tube for only 5 times Do not vortex after addition of LYS BAC Use larger tubes or reduce culture volumes for easier mixing Low purity A260 A280 lt 1 8 or gt 2 0 NucleoBond Xtra BAC Column Filter was not removed before second washing step Protein content too high due to inaccurate washing 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 Xtra Column Filter to avoid SDS carry over Only minimal amounts of DNA were loaded onto the column Excessive free binding capacity requires more extensiv
14. leads to different migration speeds of DNA larger than 30 kbp Alternatively the DNA can be restricted for example with BamHI EcoRI or Hindlll and analyzed on a 0 7 TAE agarose gel The large construct DNA with only few restriction sites show a ladder of distinct bands whereas the genomic DNA might be visible as a continuous smear in the background 4 15 Convenient stopping points Cell pellets can easily be stored for several months at 20 C Cleared lysates can be kept on ice or at 4 C for several days For optimal performance the column purification should not be interrupted However the columns can be left unattended for several hours since the columns do not run dry causing only small losses in DNA yield The eluate can be stored for several days at 4 C Note that the eluate should be warmed up to room temperature before precipitating the DNA to avoid co precipitation of salt MACHEREY NAGEL 03 2014 Rev 02 17 Large construct DNA purification 5 Storage conditions and preparation of working solutions Storage conditions and preparation of working solutions All kit components can be stored at room temperature 18 25 C and are stable for at least two years Before you start any NucleoBond Xtra BAC purification prepare the following Dissolve the lyophilized RNase A of one vial by the addition of 1 mL of Buffer RES BAC Wearing gloves is recommended Pipette up and down until the RNase A is disso
15. literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 0 24 21 969 270 e mail tech bio mn net com MACHEREY NAGEL 03 2014 Rev 02 35
16. range By rule of thumb 1 liter of E coli culture with an ODgo0 of 1 consists of 1 x 10 cells and yields about 1 5 1 8 g cell wet weight Overnight cultures containing a large construct and grown in LB medium usually reach an ODsoo of 3 under vigorous shaking in flasks The expected DNA yield for a large construct is approximately 30 40 ug per gram cell wet weight As a consequence it is important to adjust the cell mass rather than the culture volume for the best large construct purification results However since the cell mass or cell wet weight is tedious to determine it was replaced in this manual by the mathematical product of optical density at 600 nm OD and culture volume Vol two variables that are much easier to measure ODV ODgo0 X Vol mL Please note that for a correct OD determination the culture samples have to be diluted if the ODgo9 exceeds 0 5 allowing it to increase proportionally with cell mass For a well grown E coli culture a 1 10 dilution with fresh culture medium is recommended The measured ODs is then multiplied with the dilution factor 10 to result in a theoretical OD value This ODsoo is used to determine the appropriate culture volume Table 2 shows recommended ODVs along with the corresponding pairs of OD and culture volume that can be easily handled using the standard kit protocol lysis buffer volumes For example if the ODgo9 of your E coli culture is 2 use 750 mL culture for your BAC prepar
17. A can be ordered separately see ordering information If less than the recommended amount of cells is to be used e g due to bad cell growth or limited culture volumes less lysis buffer volumes can be used than given in the standard protocol 60 mL each Calculate the necessary amount according to the above given formula Note that the yield might then be significantly lower than 200 ug By using sufficient amounts of lysis buffer lysis time can be limited to 3 4 minutes and should not exceed 5 minutes Prolonged exposure to alkaline conditions can irreversibly denature and degrade especially large construct DNA and liberate contaminating chromosomal DNA into the lysate MACHEREY NAGEL 03 2014 Rev 02 13 Large construct DNA purification 4 8 Difficult to Iyse strains For large construct purification of Gram positive bacteria or strains with a more resistant cell wall it might be advantageous to start the preparation with a lysozyme treatment Proceed to resuspend the cell pellet in Buffer RES BAC containing 2 mg mL lysozyme and incubate at 37 C for 30 minutes Then follow the lysis procedure according to the NucleoBond Xtra BAC standard protocol 4 9 Setup of NucleoBond Xtra BAC Columns Ideally the NucleoBond Xtra Columns are placed into a NucleoBond Xtra Combi Rack see ordering information They are held either by the collar ring of the cartridges or by the Plastic Washers included in the kit to individua
18. L 03 2014 Rev 02 Large construct DNA purification 3 About this user manual Section 4 provides you with a detailed description of the NucleoBond Xtra BAC purification system and important information about cell growth cell lysis and the subsequent purification steps In addition sections 5 and 6 inform you about storage buffer preparation and safety instructions First time users are strongly advised to read these chapters thoroughly before using this kit Experienced users can directly proceed with the purification protocol section 7 or just use the Protocol at a glance for a quick reference Each procedural step in the purification protocol is arranged like the following example taken 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 until precipitate is completely dissolved Then proceed to cool the buffer down to room temperature 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 the suspension Incubate the mixture at room temperature 18 25 C for 5 min Warning Prolonged exposure to alkaline conditions can irreversibly denature and degrade large construct DNA and liberate contaminating chromosomal DNA into the Iysate
19. Xtra BAC Column Filter thus avoiding clogging of the filter 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 The lysate is simultaneously cleared and loaded onto the column Refill the filter if more lysate has to be loaded than the filter is able to hold Allow the column to empty by gravity flow 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 MACHEREY NAGEL 03 2014 Rev 02 23 NucleoBond Xtra BAC 10 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 Apply the buffer to the funnel shaped rim of the filter and make sure it is washing out the lysate remaining in the filter Omitting this step or just pouring the buffer directly inside the funnel may reduce plasmid yield 15 mL 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 r
20. asily dissolved in any suitable buffer e g low salt buffer or water for further use 4 2 NucleoBond Xtra BAC anion exchange columns NucleoBond Xtra BAC is a patented silica based anion exchange resin developed by MACHEREY NAGEL It is developed for routine separation of different classes of nucleic acids such as oligonucleotides RNA plasmids and large constructs like P1 BAC or PAC NucleoBond Xtra BAC Silica Resin consists of hydrophilic macroporous silica beads functionalized with MAE methyl amino ethanol The dense coating of this functional group provides a high positive charge density under acidic pH conditions As a result this permits the negatively charged phosphate backbone of plasmid DNA to bind with high specificity Figure 1 anion exchanger CH3 group MAE si NH Spacer re NN on CH 9 cz 5 NM ra eu DNA backbone Figure 1 lonic interaction of the positively charged methyl hydroxyethyl amino group with the negative phosphate oxygen of the DNA backbone In contrast to the widely used DEAE diethylaminoethyl group the hydroxy group of methyl hydroxyethyl amin can be involved in additional hydrogen bonding interactions with the DNA Birnboim H C and Doly J 1979 Nucl Acids Res 7 1513 1523 8 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA purification Due to a specialized manufacturing process that is strictly controlled and monitore
21. ation Table 2 Recommended culture volumes for large constructs NucleoBond Pellet Rec Recommended culture volume for Xtra BAC wet ODV weight ODgo0 ODeo0 ODE00 ODeo0 ODsoo ODso 2 3 4 5 6 x Vol 2 89 1500 750 mL 500 mL 375 mL 300 mL 250 mL 4 6 Lysate neutralization and LyseControl Proper mixing of the Iysate with Neutralization Buffer NEU BAC is of utmost importance for complete precipitation of SDS protein and genomic DNA Incomplete neutralization leads to reduced yields slower flow rates and potential clogging of the NucleoBond Xtra Column Filter However released plasmid DNA and especially large BAC DNA is very vulnerable at this point and shaking too much or too strongly will damage the DNA 12 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA purification Therefore do not vortex or shake but just invert the mixture very gently until a fluffy off white precipitate has formed and the LyseControl has turned colorless throughout the Iysate without any traces of blue color 4 7 Cell lysis The bacterial cell pellet is resuspended in Buffer RES BAC and lysed by a sodium hydroxide SDS treatment with Buffer LYS BAC Proteins as well as chromosomal and large construct DNA are denatured under these conditions In addition RNA is degraded by DNase free RNase A Large construct lysates have to be treated even more carefully than plasmid DNA lysates
22. can be calculated from its UV absorbance at 260 nm where an absorbance of 1 1 cm path length is equivalent to 50 ug DNA mL Note that the absolute measured absorbance should lie between 0 1 and 0 7 in order to be in the linear part of Lambert Beer s law Dilute your sample in the respective buffer if necessary The plasmid purity can also be checked by UV spectroscopy A ratio Of Asso Aaso between 1 80 1 90 and an Asgo Aaz around 2 0 indicates pure plasmid DNA An Aggo Azgo ratio above 2 0 is a sign for too much RNA in your preparation and an Asago Aaso ratio below 1 8 indicates protein contamination Note that agarose gels can resolve DNA of different sizes only to an upper limit of 20 30 kbp A a result standard agarose gels are not suitable to assess the quality of BAC DNA Large constructs and genomic DNA fragments run more or less atthe same speed and will appear in one broad band Sometimes additional slower running bands can be observed or DNA is stuck in the gel slot and does not run at all These are mostly conformational isomers or DNA that was denatured during the purification This DNA is usually large construct DNA or the same mix of large construct and genomic DNA as the main band and does not indicate a very high contamination with genomic DNA Large constructs like BAC can be distinguished from genomic DNA on agarose gels only by Pulse Field Gel Electrophoresis This system has a constantly changing electrical field that
23. cleic acids carry more charges than smaller ones and double stranded DNA carries more than single stranded RNA MACHEREY NAGEL 03 2014 Rev 02 9 Large construct DNA purification 4 3 Growth of bacterial cultures Yield and quality of large construct DNA highly depend on several factors type of culture media and antibiotics the bacterial host strain the type of DNA size and copy number but also on the growth conditions For large constructs like BAC PAC P1 or cosmid DNA LB Luria Bertani medium is strongly recommended The cell culture should be grown to an ODgoo of 1 5 2 0 to prevent starvation of the cells and degradation of large constructs Therefore incubate at 32 37 C preferably 12 16 hours overnight Use flasks of at least three or four times 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 or CircleGrow can be used In this case bacteria grow faster reaching the stationary phase much earlier than in LB medium lt 12 h and reach higher cell masses However this does not necessarily yield more DNA Overgrowing a culture might lead to a higher percentage of dead or starving cells and the resulting BAC PAC P1 or cosmid DNA might be partially degraded or contaminated with chromosomal DNA To find the optimal culture conditions the culture medium and incubation times ha
24. d the NucleoBond Xtra BAC silica beads are uniform in diameter and contain particularly large pores These special properties allow optimized flow rates and sharp well defined elution profiles NucleoBond Xtra BAC can separate distinct nucleic acid species from each other as well as proteins carbohydrates and other unwanted cellular components over a broad range of salt concentrations Figure 2 All contaminants from proteins to RNA are washed from the column Then 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 buffer 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 Plasmid DNA large constructs o Single stranded DNA Double stranded DNA ll mRNA 16S 23S rRNA m 5S rRNA Compound class Oo tRNA p Proteins dyes polysaccharides metabolites trinucleotides rRNA large constructs Plasmid DNA Absorbance at 260 nm 0 0 5 1 1 5 Salt concentration for elution M KCI Figure 2 Elution profile of NucleoBond Xtra BAC Silica Resin at pH 7 0 The more interactions a nucleic acid can form between the phosphate backbone and the positively charged resin the later it is eluted with increasing salt concentration Large nu
25. e washing Add an additional washing step with Buffer WASH BAC Reduce lysis time lt 5 min 30 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA Purification Problem Possible cause and suggestions No nucleic acid pellet formed after precipitation Pellet was lost Handle the precipitate with care Decant solutions carefully or even better pipette off the supernatant Determine DNA yield in Buffer ELU BAC in order to calculate the amount of large construct DNA that should be recovered after precipitation Large construct 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 ideally for several hours 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 Co precipitation of salt Check isopropanol purity and perform precipitation at room ni acid temperature 18 25 C but making sure to centrifuge at 4 C Do not let the eluate drip from the column into isopropanol Opagusor instead add isopropanol to the final eluate and mix immediately white instead of clear and Dissolve the pellet in water or TE buffer Precipitate DNA again glassy by adding 1 10 volume of 3 M sodium acetate pH 5 0 and 0 4 volumes of isopropanol Proceed wi
26. e NucleoBond Xtra BAC Column Filters MACHEREY NAGEL 03 2014 Rev 02 21 NucleoBond Xtra BAC 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 For an efficient cell lysis it is important that no clumps remain in the suspension 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 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 until precipitate is completely dissolved Then proceed to cool the buffer down to room temperature 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 the suspension Incubate the mixture at room temperature 18 25 C for 5 min Warning Prolonged exposure to alkaline conditions can irreversibly denature and degrade large construct DNA and liberate contaminating chromosomal DNA into 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 ly
27. emove the column filter before applying Buffer Wash BAC to avoid low purity 45 mL 12 Elution Buffer ELU BAC Heat Elution Buffer ELU BAC to 65 70 C Remove the waste container and place a 15 mL or 50 mL centrifuge tube not provided under the column Elute the large construct DNA with hot Elution 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 24 MACHEREY NAGEL 03 2014 Rev 02 NucleoBond Xtra BAC 15 mL 13 Precipitation Add 0 4 volumes of room temperature isopropanol to precipitate the eluted large construct DNA Vortex well and let the mixture sit for 2 minutes Centrifuge at 5 000 x g for 15 min at s room temperature preferably at 15 000 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
28. er for a second time with Wash Buffer WASH BAC This ensures highest yields with best achievable purity 4 12 Elution of large construct DNA Elution is carried out under high salt conditions and by a shift of pH from 6 3 to 8 5 Under these alkaline conditions the positive charge of the anion exchange resin is neutralized and large construct DNA is released To facilitate the dissociation of large construct DNA from the resin ELU BAC should be heated to 65 70 C The elution efficiency can be increased further by preventing the elution buffer from cooling down too fast 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 For any downstream application it is necessary to precipitate the DNA and to remove salt and all traces of alcohol since they disturb or inhibit enzymatic activity needed for restriction 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 4 volumes of isopropanol To prevent co precipitation of salt use room temperature 18 25 C isopropanol only and do not let the large construct DNA solution drop into a vial with isopropanol Instead add the isopropanol to the final eluate and mix immediately DNA is then collected by centrifugation and washed with 70
29. f the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for IN VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressiy marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specificat
30. holen rztliche Hilfe hinzuziehen If experiencing respiratory symptoms Call a POISON CENTER or doctor phy sician Bei Symptomen der Atemwege GIFTINFORMATIONSZENTRUM oder Arzt anrufen Wash contaminated clothing before reuse Kontaminierte Kleidung vor erneutem Tragen waschen Absorb spillage to prevent material damage Versch ttete Mengen aufnehmen um Materialsch den zu vermeiden Store in a well ventilated place Keep cool K hl an einem gut bel fteten Ort aufbewahren Store in a corrosive resistant container with a resistant inner liner In korrosionsbest ndigem Beh lter mit korrosionsbest ndiger AUskleidung aufbe wahren For further information please see Material Safety Data Sheets www mn net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenblattern www mn net com 20 MACHEREY NAGEL 03 2014 Rev 02 NucleoBond Xtra BAC 7 NucleoBond Xtra BAC purification Prepare a starter culture Inoculate a 3 5 mL starter culture of LB medium with a single colony picked from a freshly streaked agar plate Make sure that plate and liquid culture contain the appropriate selective antibiotic to guarantee large construct propagation see section 4 3 for more information Shake at 32 37 C and 300 rpm for 8 h Prepare a large overnight culture Inoculate two 250 mL overnight cultures in 1000 mL Erlenmeyer flasks by diluting the starter culture 1 1000 into the given vol
31. ions and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is 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 to get an extra copy There is no warranty for and MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or 34 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA purification components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY
32. lly adjust the height of each column see Figure 3 The Plastic Washers can also be used to hold the columns on top of suitable collection tubes or flasks The NucleoBond Xtra Combi Rack can be used in combination with NucleoBond PC 100 500 and 2000 as well Note that the NucleoBond Xtra Midi Columns can also be placed in the NucleoBond Rack Large REF 740563 A B Figure 3 Setup of NucleoBond Xtra Columns with the NucleoBond Xtra Combi Rack A Setup for clarification loading and first washing step B Setup for elution 14 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA purification 4 10 Filtration and loading of the Iysate After the alkaline lysis the sample has to be cleared from cell debris and precipitate to ensure high plasmid purity and a fast column flow rate This is achieved by passing the solution through a NucleoBond Xtra BAC Column Filter which is provided already inserted into the NucleoBond Xtra BAC Column NucleoBond Xtra BAC a NucleoBond Xtra BAC Column Filter NucleoBond Xtra BAC Column The NucleoBond Xtra BAC Column Filters are designed to eliminate the cen trifugation step after the alkaline lysis They are pre wet during column equilibration and allow a time saving simultaneous clearing of bacterial lysate and loading of the NucleoBond Xtra BAC Column Compared to lysate clearing by centrifugation or syringe filters the NucleoBond Xtra BAC Column Filter av
33. lved completely Transfer the RNase A solution back to the bottle containing Buffer RES BAC and shake well Note the date of RNase A addition The final concentration of RNase A is 100 ug mL Buffer RES BAC Store Buffer RES BAC with RNase A at 4 C The solution will be stable at this temperature for at least 6 months Buffer LYS BAC should be stored at room temperature 18 25 C since the containing SDS might precipitate at temperatures below 20 C If precipitation is observed incubate the bottle for several minutes at about 30 40 C and mix well until the precipitate is completely redissolved REF 740436 25 contains 2 x 100 mg of RNase A Make sure to dissolve RNase A of both vials each in 1 mL of Buffer RES BAC and transfer the solution back into the bottle containing Buffer RES BAC 18 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA purification 6 Safety instructions The following components of the NucleoBond Xtra BAC kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features need not be labeled with H and P phrases up to 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstoff GHS Symbol H S tze P S tze LYS BAC Sodium hydroxide lt
34. oids shearing of large DNAconstructs such as PACs or BACs by the gentle depth filter effect filtration occurs on the surface of the filter as well as inside the filter matrix Its special material and design lead to a very rapid passage of the lysate through the filter and even very large lysate volumes can be applied without the risk of clogging This is especially important for low copy plasmid purification However if more than the recommended cell mass see section 4 5 Table 2 was lysed it might be advantageous to remove the precipitate by centrifugation 10 min 5000 x g before applying the lysate to the column filter 4 11 Washing of the column The high salt concentration of the lysate prevents proteins and RNA from binding to 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 filter in two subsequent washing steps First apply the Equilibration Buffer EQU BAC to the funnel rim of the filter to wash all residual lysate out of the filter and onto the column Do not just pour the buffer inside the filter Then pull out and discard the column filter or remove the filter by turning the column upside down It is essential to wash the NucleoBond Xtra BAC Column MACHEREY NAGEL 03 2014 Rev 02 15 Large construct DNA purification without the filt
35. ructs giving poor results in downstream applications like enzymatic restriction or sequencing When noticing problems with host strains like Top10 HB101 or its derivatives like TG1 JM100 a change to DH5a or XL1 Blue should be considered The type of large construct especially the size and the origin of replication ori has a crucial influence on DNA yield In general the larger the construct or the cloned insert is the lower the expected DNA yield is due to a lower copy number Cosmids or BACs for example are maintained at copy numbers lt 20 and down to only 1 whereas vectors based on for example pUC pBluescript or pGEM can be present in several hundred copies per cell Thus all the above mentioned factors should be taken into consideration if a particular DNA yield has to be achieved Culture volume and lysis procedures have to be adjusted accordingly The prep to prep variation of yield varies much more compared to high copy plasmid purification 4 4 Chloramphenicol amplification of low copy plasmids To dramatically increase the low copy number of pMB1 colE1 derived plasmids grow the cell culture to mid or late log phase OD o0 0 6 2 0 under selective conditions with an appropriate antibiotic Then add 170 ug mL chloramphenicol and continue to incubate for an additional 8 12 hours Chloramphenicol inhibits host protein synthesis and as a result prevents replication of the host chromosome Plasmid replication however i
36. s independent of newly synthesized proteins and continues for several hours until up to 2000 3000 copies per cell are accumulated Alternatively the cell culture can be grown with only partial inhibition of protein synthesis under low chloramphenicol concentrations 10 20 ug mL resulting in a 5 10 fold greater yield of plasmid DNA Both methods show the positive side effect of much less genomic DNA per plasmid but they obviously work only with plasmids that do not carry the chloramphenicol resistance gene Furthermore the method is only effective with low copy number plasmids under stringent control e g PBR322 All modern high copy number plasmids e g pUC are already under relaxed control due to mutations in the plasmid copy number control genes resulting in an insignificant additional increase to their copy number Maniatis T Fritsch EF Sambrook J Molecular cloning A laboratory manual Cold Spring Harbor Cold Spring New York 1982 Frenkel L Bremer H Increased amplification of plasmids pBR322 and pBR327 by low concentrations of chloramphenicol DNA 5 539 544 1986 MACHEREY NAGEL 03 2014 Rev 02 11 Large construct DNA purification 4 5 Culture volume for large constructs Due to the influence of growth media TB CircleGrow 2xYT growth conditions shaking temperature incubation time and host strain or type of insert etc the final amount of cells in a bacterial culture can vary over a wide
37. se incubation after neutralization addition of Buffer NEU BAC DNA is not completely dissolved Use more TE buffer or H O to dissolve large construct DNA pellets after precipitation 32 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA purification 8 2 Ordering information Product REF Pack of NucleoBond Xtra BAC 740436 10 10 preps 740436 25 25 preps NucleoBond Xtra Combi Rack 740415 1 NucleoBond Xtra BAC Buffer Set 740437 1 Buffer RES BAC LYS BAC NEU BAC RNase A Buffer RES BAC TE 740440 1000 mL Buffer LYS BAC 740441 1000 mL Buffer NEU BAC 740442 1000 mL Buffer EQU BAC 740443 1000 mL Buffer WASH BAC 740444 1000 mL Buffer ELU BAC 740445 600 mL RNase A 740505 50 50 mg 740505 100 mg Visit www mn net com for more detailed product information MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA purification 8 3 Product use restriction warranty NucleoBond Xtra BAC kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet o
38. sis 60 mL Equilibration Buffer EQU BAC Equilibrate a NucleoBond Xtra BAC Column together with the inserted column filter with Equilibration Buffer EQU BAC Apply the buffer onto the rim of the column filter as shown in the picture and make sure to wet the entire filter Allow the column to empty by gravity flow The column does not run dry 22 MACHEREY NAGEL 03 2014 Rev 02 NucleoBond Xtra BAC 30 mL 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 The flask or tube used for this step should not be filled more than two thirds to allow 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 suspension of off white flocculate In addition the LyseControl should turn completely colorless without any traces of 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 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
39. th the precipitation protocol in this manual Pellet was over dried Nucleic Try to dissolve at higher temperatures for a longer period of time e g 2 h at 37 C or overnight at RT preferably under acid pellet constant spinning 3D shaker does not resuspend in buffer Co precipitation of salt or residual alcohol Wash the pellet again with 70 ethanol or increase the recon stitution buffer volume MACHEREY NAGEL 03 2014 Rev 02 31 Large construct DNA purification Problem Possible cause and suggestions Purified large construct 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 Precipitate DNA again by adding 1 10 volume of 3 M sodium acetate pH 5 0 and 0 4 volumes of isopropanol Proceed with the precipitation protocol in this manual and dry DNA pellet completely DNA is degraded Use only wide bore pipette tips with a large opening for dissolving large constructs to avoid shearing of the DNA For example cut off 1 cm of the pipette tip to increase the opening Make sure that all your equipment pipettes centrifuge tubes etc is clean and nuclease free Do not lyse the sample with Buffer LYS BAC for more than 5 min DNA is not completely re hybridized Increa
40. to avoid nicking and irreversible denaturation of the large constructs As a consequence shaking or vortexing of the lysate must be avoided Neutralization Buffer NEU BAC containing potassium acetate is then added to the lysate causing SDS to precipitate as KDS potassium dodecyl sulfate and pull down proteins chromosomal DNA and other cellular debris The potassium acetate buffer also neutralizes the lysate Circular DNA can revert to its native super coiled structure and remains in solution The NucleoBond Xtra BAC buffer volumes given in the standard protocol are ad justed to ensure optimal lysis for culture volumes given in section 4 5 Table 2 Using too much cell material leads to inefficient cell lysis and precipitation which can poten tially reduce large construct yield and purity Therefore lysis buffer volumes should be increased 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 ODgo9 25 Note This formula differs from the one for plasmid DNA to ensure maximum yield for large constructs For example if 500 mL of a bacterial culture ODs 3 ODV 1500 is to be lysed the appropriate volumes of lysis buffers RES BAC LYS BAC and NEU BAC are 60 mL each If more lysis buffer is needed than is provided with the kit an additional buffer set including buffers RES BAC LYS BAC NEU BAC and RNase
41. umes of LB medium also containing the appropriate selective antibiotic Refer to section 4 5 for larger culture volumes if the cultures are known to grow poorly Grow the cultures overnight at 32 37 C and 200 250 rpm for 12 16 h 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 ODgoo of around 3 and should yield around 100 ug of large construct DNA see to section 4 5 for more information 2 x 250 mL Harvest bacterial cells Measure the cell culture ODgo9 and determine the recommended culture volume V ML 1500 ODgoo Pellet the cells by centrifugation at 4 500 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 th
42. urification M 12 34 5 M Marker 4 Hindill Cleared lysate large construct and degraded RNA 2 Lysate flow through no large construct laat w w DNA but degraded RNA 3 Wash flow through no large construct DNA or residual RNA 4 Eluate pure large construct DNA 5 EcoRI restriction Figure 4 Exemplary analytical check of NucleoBond Xtra BAC purification samples Large construct 240 kbp BAC bacterial strain E coliDH5a 20 uL of each precipitated sample has been analyzed on a 1 agarose gel Equal amounts of large construct DNA before lane 1 and after lane 4 purification using NucleoBond Xtra BAC are shown with a recovery of gt 90 MACHEREY NAGEL 03 2014 Rev 02 27 Large construct DNA purification Problem Possible cause and suggestions No or low large construct DNA yield Large construct did not propagate Check large construct content in the cleared Iysate see Figure 4 Use colonies from fresh plates for inoculation and add 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 incubate the bottle for several minutes at 30 40 C and mix well until SDS is redissolved
43. ve to be optimized for each host strain large construct combination individually Cell cultures should be grown under antibiotic selection at all times to ensure large construct DNA propagation Without this selective pressure cells tend to lose a large construct during cell division Since bacteria grow much faster without the burden of an additional chromosome they take over the culture rapidly and the yield goes down regardless of the cell mass Table 1 gives information on concentrations of commonly used antibiotics Table 1 Information about antibiotics according to Maniatis Antibiotic Stock solution Storage Working concentration concentration Ampicillin 50 mg mL in H O 20 C 50 100 pg mL Chloramphenicol 34 mg mL in EtOH 20 C 25 170 g mL Kanamycin 10 mg mL in H O 20 C 10 50 pg mL Streptomycin 10 mg mL in H O 20 C 10 50 pg mL Tetracycline 5 mg mL in EtOH 20 C 10 50 ug mL Carbenicillin 50 mg mL in H O 20 C 20 60 ug mL Maniatis T Fritsch EF Sambrook J Molecular cloning A laboratory manual Cold Spring Harbor Cold Spring New York 1982 10 MACHEREY NAGEL 03 2014 Rev 02 Large construct DNA purification The E coli host strain mostly influences the quality of the large construct DNA Strains like DH5a or XL1 Blue usually produce high quality super coiled DNA whereas strains like HB101 with high levels of endonuclease activity might yield lower quality large const
44. 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 H O to dissolve the pellet Less than 500 uL can be applied if the yield is expected to be very low 500 1000 uL Ideally incubate overnight at 4 C to dissolve BAC DNA completely Avoid pipetting up and down since large DNA constructs are prone to shearing Instead shake the tube gently and use only wide bore pipette tips with a large opening or cut the tip to increase the opening 4 C over night If the dissolved DNA pellet is very viscous add more buffer TE or HO to ensure complete dissolving and a correct quantification Determine large construct yield by UV spectrophotometry MACHEREY NAGEL 03 2014 Rev 02 25 Large construct DNA purification 8 Appendix 8 1 Troubleshooting If you experience problems with reduced yield or purity it is recommended to check which purification step of the procedure is causing the problem First the bacterial culture has to be checked for sufficient growth OD in the presence of an appropriate selective antibiotic Table 1 section 4 3 Second aliquots of the cleared Iysate the flow through the combined washing steps Buffer

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