PureLink™ HiPure Plasmid Purification Kit
Contents
1. Plasmid Number Midiprep Maxiprep High copy number 200 uL 500 uL Low copy number 100 pL 200 uL 8 Optional Centrifuge the DNA in TE Buffer TE for 1 minute at room temperature at high speed to remove any insoluble particles that may be present Transfer the supernatant containing the DNA into a fresh tube 9 Store the purified DNA at 20 C or proceed to the downstream application Kit Contents and Storage Types of This manual is supplied with the kits listed below Products Product Quantity Catalog no PureLink HiPure Plasmid Filter Midiprep Kit 25 preps K2100 14 50 preps K2100 15 PureLink HiPure Plasmid Filter Maxiprep Kit 10 preps K2100 16 25 preps K2100 17 PureLink HiPure Plasmid FP Filter and 10 preps K2100 26 Precipitator Maxiprep Kit 25 preps K2100 27 Shipping and All components of the PureLink HiPure Plasmid Filter Storage Purification Kits are shipped at room temperature Upon receipt store all components at room temperature TM Contents The components included in the PureLink HiPure Plasmid Filter Purification Kits are listed below See next page for buffer composition Component Midiprep Maxiprep FP K2100 14 K2100 15 K2100 16 K2100 17 K2100 26 K2100 27 Resuspension Buffer 250mL 500 mL 100 mL 250 mL 100 mL 250 mL R3 RNase A 15mL 28mL 650pL 15mL 650pL 15mL Lysis Buffer L7 250mL 500mL 100mL 250mL 100mL 250mL Precipitation butter 22. Specifications and results are based on high copy number plasmids Binding capacity depends on plasmid copy number type and size and volume of bacterial culture used DNA yield depends on plasmid copy number type and size bacterial strain and growth conditions Plasmid DNA isolated using the PureLink HiPure Plasmid Filter Purification Kits is suitable for a wide variety of downstream applications such as e Mammalian transfections e Automated fluorescent DNA or manual sequencing e PCR e Cloning in vitro transcription e Restriction digestion Experimental Overview Introduction MidiPrep gt art qu mg KE i ag 2a Ga 9 1 I amm 1 hf emm e CIT 6 The flow chart below illustrates the steps for isolating plasmid DNA from bacteria using the PureLink HiPure Plasmid Filter Purification kits 15 ml Equilibration Buffer EQ1 Harvest Cells Y 10 ml Resuspension Buffer R3 Y 10 ml Lysis Buffer L7 Y 10 ml Precipitation Buffer N3 Y Load Filter Column Y Clear Lysate by Filtration Y 20 ml Wash Buffer W8 Y 5 ml Elution Buffer E4 Y 3 5 ml Isopropanol x 3 ml 70 Ethanol Y 200 ul TE Buffer MaxiPrep CE g ap Mg C quum cr 10MII T Ga ii IG a am 30 ml Equilibration Buffer EQ1 Harvest Cells v 10 ml Resuspension
3. 3 Add 10 mL Resuspension Buffer R3 with RNase A to the cell pellet in the tube and resuspend the cells Gently shake the tube until cell suspension is homogeneous 4 Add 10 mL Lysis Buffer L7 Place the cap on the tube and ensure it is secure Mix gently by inverting the capped tube until the lysate mixture is thoroughly homogenous Do not vortex 5 Incubate the lysate at room temperature for 5 minutes Do not exceed 5 minutes 6 Add 10 mL Precipitation Buffer N3 and mix immediately by inverting the tube until the mixture is thoroughly homogeneous Do not vortex 7 Proceed to Loading Filter Column and Washing DNA next page Continued on next page Midiprep Procedure Continued Loading Filter 1 Column and Washing DNA Eluting DNA 1 Transfer the precipitated lysate from Step 6 previous page including all the precipitated material into the equilibrated HiPure Filter Midi Column Let the lysate pass through the filter by gravity flow until the flow stops 10 15 minutes or becomes very slow lt 1 drop per 10 seconds Discard the flow through Optional The final DNA yield may be increased by washing the residual bacterial lysate in the HiPure Filter Midi column with 10 mL Wash Buffer W8 Again let the buffer flow through the HiPure Filter Midi Column by gravity flow until the flow stops or dripping becomes very slow Immediately after the HiPure Filter Midi Column has stopped dripping remove and discard
4. 4 Add Wash Buffer W8 to the filter column Midiprep Maxiprep 20mL 50 mL 5 Allow wash buffer to flow through the filter column by gravity flow Discard the flow through Continued on next page Experienced Users Procedure Continued Step Eluting DNA Precipitating DNA Action 1 Place a sterile 15 mL Midi or 50 mL Maxi centrifuge tube elution tube under the HiPure filter column 2 Add Elution Buffer E4 to the column to elute the DNA Allow solution to drain by gravity flow Midiprep Maxiprep 5mL 15mL 3 The elution tube contains the DNA Discard the HiPure Filter Column Before starting If you are using the PureLink HiPure Plasmid FP Maxiprep kit and you are precipitating the DNA using the PureLink HiPure Precipitator Module refer to the precipitator protocol provided on page 18 at this time To precipitate DNA using centrifugation follow the steps below 1 Add isopropanol to the elution tube containing the DNA Midiprep Maxiprep 3 5 mL 10 5 mL 2 Incubate DNA with isopropanol for 2 minutes at room temperature 3 Centrifuge the tube for at gt 12 000 x g for 30 minutes at 4 C Remove and discard the supernatant 4 Add 70 ethanol to resuspend the pellet in the tube Midiprep Maxiprep 3mL 5mL 5 Centrifuge the tube for at 212 000 x g for 5 minutes at 4 C Remove and discard the supernatant 6 Air dry the pellet for 10 minutes 7 Add TE Buffer TE to the tube to resuspend the pellet
5. Buffer R3 Y 10 ml Lysis Buffer L7 M 10 ml Precipitation Buffer N3 Y Load Filter Column Y Clear Lysate by Filtration Y 50 ml Wash Buffer W8 Y 15 ml Elution Buffer E4 v 10 5 ml Isopropanol h 5 ml 70 Ethanol Y 500 ul TE Buffer MaxiPrep FP D D fa a fa g p g T G H m By V 30 ml Equilibration Buffer EQ1 Harvest Cells Y 10 ml Resuspension Buffer R3 Y 10 ml Lysis Buffer L7 Y 10 ml Precipitation Buffer N3 Y Load Filter Column M Clear Lysate by Filtration M 50 ml Wash Buffer W8 Y 15 ml Elution Buffer E4 Y 10 5 ml Isopropanol Y Collect DNA using HiPure Precipitator Y 0 75 1 0 ml TE Buffer Methods Before Starting Introduction Bacterial Cultures Plasmid Type and Copy Number Review the information in this section before starting Guidelines are included for growing the overnight bacterial cell culture and for determining the appropriate amounts of starting material based on the plasmid copy number used Some of the buffers in the PureLink HiPure Plasmid Filter Purification Kits contain hazardous chemicals For your protection always wear a laboratory coat disposable gloves and eye protection when handling the buffers Grow transformed E coli cells overnight in LB Luria Bertani medium with th
6. Continued Problem Cause Solution Precipitator is Too much DNA Only load the eluate from one anion clogged FP Applied exchange column onto the Maxiprep Kit precipitator Using eluate from more than one will overload the membrane DNA precipitated Ethanol precipitated DNA consists of with ethanol instead fine particles that may clog the of isopropanol precipitator Always use isopropanol to precipitate plasmid DNA Additional Plasmid DNA Incubate the lysate in Lysis Buffer plasmid forms permanently L7 at room temperature for no present denatured band longer than 5 minutes migrating faster than supercoiled DNA RNA Lysate at improper e Carefully remove all medium contamination pH salt before resuspending cells concentration or e Make sure not to add an excess temperature of Precipitation Buffer N3 when neutralizing the lysate Lysate left on Filter Once the lysate is loaded onto the Column too long column avoid delays in processing Lysate droplets remained on walls of column at elution Wash droplets of lysate from the walls of the Filter Column with the Wash Buffer W8 RNase A digestion e Make sure RNase A is added to incomplete Resuspension Buffer R3 e Use recommended volume of Resuspension Buffer e Ensure the Resuspension Buffer with RNAse A is stored at 4 C e If necessary increase RNase A concentration to 400 ug ml Slow filtration Used high culture e Reduce volume of c
7. experiments with PureLink HiPure Plasmid Filter Purification Kits Problem Cause Solution Low plasmid Buffers not stored e Store Lysis Buffer L7 and DNA yield correctly Equilibration Buffer EQ1 at room temperature e Store Resuspension Buffer R3 with added RNase A at 4 C Low copy number Increase the volume of starting plasmid culture Carefully remove all medium before resuspending cells Doubling the volumes of the Resuspension Buffer R3 Lysis Buffer L7 and Precipitation Buffer N3 may help to increase plasmid yield and quality Lysate has improper Make sure that the correct volume of pH or salt Precipitation Buffer N3 is added concentration to bind when neutralizing the lysate column Plasmid DNA pellet Do not dry the DNA pellet with a over dried vacuum system Air dry the DNA pellet Precipitator e Attach the precipitator to the membrane damaged syringe nozzle using the luer resulting in leaks FP lock mechanism without Maxiprep Kit applying excessive force e Prior to removing the plunger from the syringe always remove the precipitator to avoid damaging the membrane e Do not apply excessive pressure while pushing the solution through the precipitator Genomic DNA Genomic DNA Gently invert tubes to mix after contamination sheared during adding buffers Do not vortex as it handling can shear genomic DNA 23 Continued on next page Troubleshooting
8. the inner Filtration Cartridge from the column Note Do not reuse the Filtration Cartridge The cartridge is designed for single use only Wash the Midi column with 20 mL Wash Buffer W8 Allow the solution in the column to drain by gravity flow Discard the flow through Proceed to Eluting DNA below Place a sterile 15 mL centrifuge tube elution tube under the HiPure Filter Midi column Add 5 mL Elution Buffer E4 to the Midi column to elute the DNA Allow the solution to drain by gravity flow Do not force out any remaining solution The elution tube contains the purified DNA Discard the HiPure Filter Midi column Proceed to Precipitating DNA next page For DNA precipitation using the Midiprep Kit you can use Note the PureLink HiPure Precipitator Module page 25 which allows DNA precipitation within 10 minutes without centrifugation or you can follow the protocol for Precipitating DNA with Isopropanol next page to perform traditional DNA precipitation using centrifugation Refer to the manual supplied with the PureLink HiPure Precipitator Module for a detailed protocol Continued on next page 12 Midiprep Procedure Continued Precipitating DNA with Isopropanol Storing DNA 13 1 Add 3 5 mL isopropanol to the elution tube containing the DNA see Eluting DNA page 12 Mix well Incubate the DNA isopropanol mixture for 2 minutes at room temperature Centrifuge the tube at gt 12
9. 000 x g for 30 minutes at 4 C Carefully remove and discard the supernatant Resuspend the DNA pellet in 3 mL 70 ethanol Centrifuge the tube at gt 12 000 x g for 5 minutes at 4 C Carefully remove and discard the supernatant Air dry the pellet for 10 minutes Resuspend the DNA pellet in 200 uL TE Buffer TE for high copy number plasmids For low copy number plasmids use 100 pL TE Buffer TE Note Occasionally insoluble particles may be present These particles do not influence the quality of the DNA and can be easily removed To remove insoluble particles centrifuge the DNA solution at high speed for 1 minute at room temperature Transfer the supernatant DNA sample into a fresh tube Store the purified DNA at 20 C or proceed to the desired downstream application Note To avoid repeated freezing and thawing of DNA store the purified DNA at 4 C for immediate use or aliquot the DNA and store at 20 C for long term storage Continued on next page Maxiprep Procedure Introduction Before Starting Materials Needed Components Supplied with the Kit Note The PureLink HiPure Plasmid DNA Maxiprep Kit allows purification of 500 850 ug of high quality plasmid DNA from 100 200 mL overnight E coli cultures in 2 hours when cloning high copy number plasmids Verify that the Resuspension Buffer R3 contains RNase A and no precipitate has formed in the Lysis Buffer L7 See page 9 for details e Ov
10. 50mL 500mL 100mL 250mL 100mL 250mL N3 ee ed Buffer 375 mL 2x400mL 300mL 2x400mL 300mL 2x400mL Wash Buffer W8 2 x 400 mL 3 x 500 mL 2 x 300 mL 3 x 500 mL 2 x 300 mL 3 x 500 mL Elution Buffer E4 125 mL 250 mL 250 mL 400 mL 250 mL 400 mL TE Buffer TE 15 mL 15 mL 15 mL 30 mL 15 mL 30 mL HiPure Filter Columns 25 each 50 each 10 each 25 each 10 each 25 each Column Holders 5 each 10 each 3 each 5 each 3 each 5 each PureLink HiPure Precipitator Module See the manual included with the PureLink HiPure Precipitator Module for detailed contents and protocol 10 each 25 each Continued on next page vi Kit Contents and Storage Continued Buffer The composition of buffers included in the PureLink Composition HiPure Plasmid Filter Purification Kits is listed below Buffer Composition Resuspension Buffer R3 50 mM Tris HCl pH 8 0 10 mM EDTA RNase A 20 mg ml in Resuspension Buffer R3 Lysis Buffer L7 0 2 M NaOH 1 w v SDS Precipitation Buffer N3 3 1 M Potassium acetate pH 5 5 Equilibration Buffer 0 1 M Sodium acetate pH 5 0 EQ1 0 6 M NaCl 0 15 v v Triton X 100 Wash Buffer W8 0 1 M Sodium acetate pH 5 0 825 mM NaCl Elution Buffer E4 100 mM Tris HCI pH 8 5 1 25 M NaCl TE Buffer TE 10 mM Tris HCl pH 8 0 0 1 mM EDTA Intended Use For research use only Not intended for human or animal diagnostic or therapeutic uses vii Introduction About the Kit Intr
11. 8 Continued on next page 16 Maxiprep Procedure Continued Note Precipitating DNA with Isopropanol Storing DNA 17 DNA precipitation can be completed using centrifugation or with the PureLink HiPure Precipitator Module included with the FP Maxiprep Kit or purchased as a separate kit see page 25 The precipitator module allows DNA precipitation within 10 minutes without any centrifugation steps Refer to the section below to precipitate DNA with isopropanol by centrifugation For DNA precipitation using the PureLink HiPure Precipitator Module refer to page 18 For a detailed protocol on using the precipitator module refer to the product insert included with the precipitator 1 Add 10 5 mL isopropanol to the DNA to the elution tube Mix well 2 Centrifuge the tube at 212 000 x g for 30 minutes at 4 C Carefully remove and discard the supernatant 3 Add5mL 70 ethanol to resuspend the DNA pellet 4 Centrifuge the tube at 212 000 x g for 5 minutes at 4 C Carefully remove and discard the supernatant Air dry the pellet for 10 minutes 6 Resuspend the DNA pellet in 500 uL TE Buffer TE For low copy number plasmids use 200 uL TE Buffer TE Note Occasionally insoluble particles may be present These particles do not influence the quality of the DNA and can be easily removed To remove insoluble particles centrifuge the DNA solution at high speed at room temperature for 1 minute Transfer the
12. any centrifugation steps This saves time and reduces the risk of losing the DNA pellet during supernatant removal To precipitate and desalt the DNA isopropanol is added to the eluted DNA and then applied to the HiPure Precipitator using a large syringe After a subsequent washing and drying step the plasmid DNA is easily eluted from the HiPure Precipitator with TE buffer Continued on next page About the Kit Continued System Overview Advantages TM The PureLink HiPure Plasmid Filter Purification Kits use a patented anion exchange resin to purify plasmid DNA to a level of purity that is equivalent to two passes through CsCI gradients The patented resin provides high binding capacity with fast flow rates high resolution high yield and efficient endotoxin removal E coli cells are harvested by centrifugation then resuspended in Resuspension Buffer R3 with RNase A and lysed with Lysis Buffer L7 Precipitation Buffer N3 is then added to the lysate The lysate is poured into a pre packed anion exchange column fitted with the Filtration Cartridge unit In one simple combined step the lysate is clarified and the negatively charged phosphates of the DNA backbone interact with the positive charges on the surface of the anion exchange resin The temperature salt concentration and pH of the solutions are optimized for efficient binding of DNA A single column wash under moderate salt conditions using Wash Buffer W8 re
13. ation from DNases e Ensure that no DNase is introduced into the sterile solutions supplied with the kit e Make sure that all equipment coming in contact with DNA is sterile including pipette tips and tubes e Use the PureLink Nucleic Acid Purification Rack for column purification see below e Perform the recommended wash steps during purification to obtain the best results e Use the TE Buffer TE provided or 10 mM Tris HCl pH 8 5 to resuspend the DNA pellet Continued on next page Before Starting Continued Purification Rack Using the Column Holder Buffers The PureLink Nucleic Acid Purification Rack see page 25 is designed specifically for use with PureLink HiPure Plasmid Filter Midiprep and Maxiprep Kits The PureLink Nucleic Acid Purification Rack consists of the following e Column Holder Rack for processing 12 miniprep 8 midiprep and 4 maxiprep columns e Collection Tube Rack capable of accommodating various types and sizes of recovery tubes e Large Capacity Waste Tray for collecting waste The Column Holders provided in the kit allow columns to be supported in an upright position when placed in the mouth of a flask To use the Column Holder slip the column through the hole in the center of the Column Holder The column with Column Holder can then be placed in the mouth of a flask Resuspension Buffer R3 Add RNase A to the Resuspension Buffer R3 according to instructi
14. control Analysis and product qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box 26 Purchaser Notification Limited Warranty 27 Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Theref
15. dilution of the DNA solution in 10 mM Tris HCL pH 7 5 Mix well Measure the absorbance at 260 nm A260 of the dilution in a spectrophotometer using a cuvette with an optical path length of 1 cm blanked against 10 mM Tris HCl pH 7 5 2 Calculate the concentration of DNA using the formula DNA ug ml A260 x 50 x dilution factor For DNA A2 1 for a 50 ug ml solution measured in a cuvette with an optical path length of 1 cm Quant iT DNA Assay Kits The Quant iT DNA Assay Kits see page 25 for ordering information provide a rapid sensitive and specific method for dsDNA quantitation with minimal interference from RNA protein ssDNA primers or other common contaminants that affect UV absorbance The kit contains a state of the art quantitation reagent pre diluted standards for standard curve and a pre made buffer The assay is designed for reading in standard fluorescent readers fluorometers or Qubit Fluorometer page 25 Typically DNA isolated using the PureLink HiPure Plasmid Filter Purification Kit has an Aeo A280 ratio gt 1 80 when samples are diluted in Tris HCl pH 7 5 indicating that the DNA is reasonably clean of proteins that could interfere with downstream applications Absence of contaminating RNA may be confirmed by agarose gel electrophoresis 20 Expected Results DNA Yield High copy number plasmid DNA was purified in triplicate from E coli TOP10 transformed with pcDNA 3 1 His LacZ usin
16. e appropriate antibiotic The bacterial culture should have a cell density of approximately 10 cells ml or an absorbance of 1 1 5 at 600 nm Acoo Use bacterial culture in transition between exponential phase and stationary phase The PureLink HiPure Plasmid Filter Purification kits allow purification of all types and sizes of plasmid DNA Use a high copy number plasmid to obtain a good yield of plasmid DNA High copy number plasmids typically yield 2 6 ug DNA ml LB culture grown overnight Typical yields from low copy number plasmids are highly dependent upon culture conditions and vector host strain combinations If you are using a low copy number plasmid you will need to use a higher volume of bacterial culture The table below lists the volumes of bacterial culture required for Midiprep and Maxiprep plasmid DNA purification depending on the plasmid copy number used Type of Plasmid Midiprep Maxiprep High copy number 15 25 mL 100 200 mL plasmid Low copy number 25 100 mL 250 500 mL plasmid Continued on next page Before Starting Continued Specific Protocols O Og EN RE NOTS l The following protocols are provided for purifying plasmid DNA using the various kits discussed in this manual Midiprep kit protocol Page 10 Maxiprep kit protocol Page 14 Follow the recommendations below to obtain the best results e Maintain a sterile environment when handling DNA to avoid any contamin
17. echnology The HiPure technology is based on anion exchange chromatography and uses a patented resin composed of small particles with a uniform pore size The HiPure technology provides high yields of highly pure plasmid DNA with reproducible performance The spacer arm with increased length provides improved DNA binding efficiency The unique patented ion exchange moiety provides high efficiency separation of DNA from cellular contaminants including RNA NH GN CH CH L CH CH Filter Columns The HiPure Filter Columns contain the Filtration Cartridge prepackaged in the DNA Binding Column The column is fitted with anion exchange resin see below This design of the HiPure Filter Column allows sample clarification by filtration and plasmid DNA binding in one combined step HiPure Filtration Cartridge Peng Filter Column Continued on next page About the Kit Continued PureLink Column Holder PureLink HiPure Precipitator Module The Column Holders in the kit allow Midi and Maxi Columns to be supported in an upright position when placed in the mouth of an Erlenmeyer or similar flask DNA Binding Column Column Holder The PureLink HiPure Precipitator Module included with the PureLink HiPure Plasmid FP Filter and Precipitator Maxiprep Kit cat nos K2100 26 and K2100 27 allows rapid isopropanol precipitation of the eluted DNA without
18. enient electrophoresis of Ladders DNA samples A large variety of DNA ladders is available from Invitrogen for sizing DNA Visit our website at www invitrogen com or contact Technical Support page 26 for more details on these products 25 Technical Support Web Visit the Invitrogen website at www invitrogen com for Resources e Technical resources including manuals vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com SDS Safety Data Sheets SDSs are available at www invitrogen com sds Certificate of The Certificate of Analysis provides detailed quality
19. ernight culture of transformed E coli cells page 7 e Isopropanol e 70 ethanol e Sterile microcentrifuge tubes e PureLink Nucleic Acid Purification Rack page 25 e Tubes or centrifuge bottles for harvesting cells e Centrifuge and rotor appropriate for harvesting cells e 50 mL centrifuge tubes capable of withstanding centrifugation forces gt 12 000 x g e Centrifuge capable of centrifuging at gt 12 000 x g at 4 C e Resuspension Buffer R3 with RNase A e Lysis Buffer L7 e Precipitation Buffer N3 e Equilibration Buffer EQ1 e Wash Buffer W8 e Elution Buffer E4 e TE Buffer TE e HiPure Filter Maxi Columns e Column Holder e PureLink HiPure Precipitator Module supplied with cat nos K2100 26 and K2100 27 only For Maxipreps of low copy number plasmids from bacterial cultures of gt 200 mL use twice the amount of Resuspension Buffer R3 Lysis Buffer L7 and Precipitation Buffer N3 as directed in the protocol next page Order the PureLink HiPure BAC Buffer kit page 25 for additional buffers if the buffers in the kit are insufficient for using all of the columns when following this protocol Continued on next page 14 Maxiprep Procedure Continued Equilibrating the Column Preparing Cell Lysate 15 The PureLink HiPure Filter Maxi Columns are pre packaged with the Filtration cartridge inserted into the column housing 1 Use the Column Holder to support a HiPure Fi
20. fer the precipitated lysate from Step 5 in Preparing Cell Lysate previous page including all the precipitated material into the equilibrated HiPure Filter Maxi Column Let the lysate run through the filter by gravity flow until the flow stops 10 15 minutes or becomes very slow 1 drop per 10 seconds Discard the flow through Optional The final DNA yield may be increased by washing the residual bacterial lysate in the HiPure Filter Maxi Column with 10 mL Wash Buffer W8 Again let the buffer flow through the HiPure Filter Maxi Column by gravity flow until the flow stops or dripping becomes very slow Immediately after the HiPure Filter Maxi Column has stopped dripping remove the inner Filtration Cartridge from the column and discard Note Use the HiPure Filtration Cartridge only once The cartridge is for single use only Wash the Maxi column with 50 mL of Wash Buffer W8 Allow the solution in the column to drain by gravity flow Discard the flow through Proceed to Eluting DNA below Place a sterile 50 mL centrifuge tube elution tube under the HiPure Filter Maxi column Add 15 mL Elution Buffer E4 to the Maxi column to elute the DNA Allow the solution to drain by gravity flow Do not force out any remaining solution The elution tube contains the purified DNA Discard the HiPure Filter Maxi column Proceed to Precipitating DNA with Isopropanol next page or Precipitating DNA Using Precipitator Module page1
21. g PureLink HiPure Plasmid Filter Purification kits as described in this manual The plasmid DNA was measured using the Quanti iT Kit page 25 DNA yield information is provided below Note The plasmid DNA yield depends on plasmid copy number type and size bacterial strain and growth conditions Kit Type Column Binding Starting Culture DNA Yield Capacity Volume Midiprep 350 ug 15 25 mL 100 350 ug Maxiprep 850 ug 100 200 mL 500 850 ug Example Plasmid DNA was isolated as described above The purified Results plasmid DNA 100 ng was analyzed on a 1 2 E Gel agarose gel The 1 Kb Plus DNA Ladder was used as marker left lane of each gel A B 12 216 12 216 8 144 8 144 Panel A PureLink HiPure Plasmid Filter Midiprep Kit Panel B PureLink HiPure Plasmid Filter Maxiprep Kit Continued on next page 21 Expected Results Continued Summary of The summary of expected results using the PureLink Expected HiPure Plasmid Filter Purification Kits is listed in the table Results below Kit Type Midiprep Maxiprep Processing Time 2 hours 2 hours Plasmid DNA Yield 100 350 pg 500 850 ug Endotoxin 0 1 1 0 EU ug 0 1 1 0 EU ug OD 260 280 1 95 1 98 Sequencing Capillary Successful Successful Restriction Enzyme Digestion Successful Successful When using pyrogen free plastic ware and glassware Appendix Troubleshooting Introduction Review the information below to troubleshoot your
22. hrough the precipitator Repeat Step 8 once Blot any ethanol droplets on the precipitator nozzle with a paper towel Detach the precipitator from the 30 mL syringe and discard the 30 mL syringe Remove a 5 mL syringe supplied with the precipitator module from the package and remove the plunger from the syringe Attach the precipitator to the 5 mL syringe To elute the plasmid DNA from the precipitator Add 0 75 1 0 mL TE buffer to the 5 mL syringe Insert the plunger and place the precipitator over a clean sterile microcentrifuge tube Push the plunger to elute the plasmid DNA into the new tube Optional Perform a second elution to maximize DNA recovery Detach the precipitator from the syringe remove the plunger and reattach the precipitator to the syringe nozzle Load the entire volume of eluate from Step 13 into the syringe Place the precipitator nozzle over a new microcentrifuge tube Insert and push the plunger to perform the second elution and elute the DNA into the microcentrifuge tube Store the eluted DNA at 20 C long term or 4 C short term or proceed to downstream application Estimating DNA Yield and Quality Introduction DNA Yield Estimating DNA Quality Once you have isolated the plasmid DNA you may determine the quantity and quality of the purified DNA as described below Perform DNA quantitation using UV absorbance at 260 nm or Quant iT DNA Assay Kits UV Absorbance 1 Prepare a
23. invitrogen by technologies PureLink HiPure Plasmid Filter Purification Kits For Midi and Maxi preparation of Plasmid DNA Catalog nos K2100 14 K2100 15 K2100 16 K2100 17 K2100 26 K2100 27 New Improved Column Design See page 3 for details Rev Date 20 June 2010 Manual part no 25 0880 MAN0000545 Table of Contents Experienced Users Procedure hone o des iv Kit Contents and Storage eee RR eee vi Luton ing E E T 1 Aboutthe Kit i eiae nU Ula ERR AERA E M RUE 1 Experimental Overview Methods niin A ci ote eceeo cte ecc ec cu e ro A Before Starting a i ipsia oido memi iudi i i Gets 7 Midiprep Procedure 6 bietet teret tette a 10 Maxiprep Proced te uere no the eam eH di 14 Estimating DNA Yield and Quality sse 20 Expect d Results e tree tetigere 21 LIB 23 Troubleshooting teen mete tete eye eig 23 Accessory PrtOGQUcts e reete rte ee et rere eredi 25 Technical Support ird rer o erae tbe es 26 Purchaser Notification eee c tete ee alpes tee Sha tee tps 27 iii Experienced Users Procedure Introduction Step Preparing Cell Lysate Loading Filter Column and Washing DNA iv This quick reference sheet is included for experienced users of the PureLink HiPure Plasmid Filter Midiprep Maxiprep and FP Maxiprep Kits If you are a first time user of these kits refer to the details in the manual Action Before Beginning App
24. lter Maxi Column in the mouth a flask see page 9 or place the Maxi Column on the PureLink Nucleic Acid Purification Rack see manual supplied with the rack for more details 2 Apply 30 mL Equilibration Buffer EQ1 directly into the Filtration Cartridge which is inserted into the Maxi Column 3 Allow the solution in the HiPure Filter Maxi Column to drain by gravity flow 4 Prepare the cell lysate see below while the HiPure Filter Maxi Column is equilibrating For high copy number plasmids use 100 200 mL of an overnight LB culture per sample For low copy number plasmids harvest 250 500 mL of an overnight LB culture per sample 2 Harvest the cells by centrifuging the overnight LB culture at 4 000 x g for 10 minutes Remove all medium 3 Add 10 mL Resuspension Buffer R3 with RNase A to the pellet and resuspend the cells until homogeneous 4 Add 10 mL Lysis Buffer L7 Mix gently by inverting the capped tube until the lysate mixture is thoroughly homogeneous Do not vortex Incubate at room temperature for 5 minutes Note Do not allow lysis to proceed for more than 5 minutes 5 Add 10 mL Precipitation Buffer N3 and mix immediately by inverting the tube until the mixture is thoroughly homogeneous Do not vortex 6 Proceed to Loading Filter Column and Washing DNA next page Continued on next page Maxiprep Procedure Continued Loading Filter 1 Column and Washing DNA Eluting DNA 1 Trans
25. ly Equilibration Buffer EQ1 to Midiprep columns 15 mL and Maxiprep columns 30 mL Verify the Resuspension Buffer R3 contains RNase A and that no precipitate has formed in the lysis Buffer L7 See page 9 for details 1 Grow bacterial culture overnight in LB medium Place appropriate amount of culture in a 50 mL disposable centrifuge tube for cell harvesting see table below Plasmid Number Midiprep Maxiprep High Copy Number 15 25 mL 100 200 mL Low Copy Number 25 100 mL 250 500 mL 2 Harvest cells by centrifugation at 4 000 x g for 10 minutes Remove all medium Note If you are using gt 200 mL culture of high copy number plasmids for the Maxiprep double the amount of Resuspension Buffer R3 with RNase A Lysis Buffer L7 and precipitation Buffer N3 for best results 3 Resuspend cell pellet in 10 mL Resuspension Buffer R3 with RNase A Gently shake until cell suspension is homogenous 4 Transfer cell suspension to a new 50 mL centrifuge tube 5 Add 10 mL Lysis Buffer L7 Mix until homogenous 6 Add 10 mL Precipitation Buffer N3 to the lysate Mix by inverting the capped tube gently 1 Transfer precipitated lysate into the HiPure Filter Midi or Maxi Column Allow lysate to filter through the column by gravity flow 2 Optional Wash residual lysate in the filter column with 10 mL Wash Buffer W8 Allow buffer to flow through column by gravity flow 3 Discard inner filtration cartridge
26. moves RNA proteins carbohydrates and other impurities while the plasmid DNA remains bound to the resin The plasmid DNA is eluted under high salt conditions with the Elution Buffer E4 The eluted DNA is desalted and concentrated by alcohol precipitation or using the PureLink HiPure Precipitator Module included with the PureLink HiPure Plasmid FP Filter and Precipitator Maxiprep Kit or available separately The entire protocol can be completed in 1 5 2 hours TM The PureLink HiPure Plasmid Filter Purification Kits offer the following advantages e Bacterial lysate clarification by a simple filter column procedure without centrifugation e No centrifugation required for alcohol precipitation when using the PureLink HiPure Precipitator Module e High quality purified plasmid DNA suited for mammalian transfections e High plasmid DNA yields with up to 350 ug for Midipreps and 850 ug for Maxipreps e Reliable performance of the purified plasmid DNA in a variety of downstream applications Continued on next page About the Kit Continued System Specifications Downstream Applications Specifications Midiprep Maxiprep Starting E coli culture 15 25 mL 100 200 mL volume Column Binding Capacity 350 ug 850 ug Filtration Cartridge 60 mL 60 mL Reservoir Capacity Column Reservoir 60 mL 60 mL Capacity Elution Volume 5mL 15 mL DNA Recovery 90 95 90 95 Expected DNA Yield 100 350 ug 500 850 ug
27. oduction Kit Formats The PureLink HiPure Plasmid Filter Purification Kits allow isolation of high yields of highly pure plasmid DNA The HiPure Filter Column provides rapid clearing of the bacterial lysate without the need for centrifugation The lysate Filtration Cartridge is integrated into the DNA Binding Column and combines the steps of clearing the bacterial lysate with binding the DNA directly to the anion exchange resin see next page The HiPure Filter Column protocol reduces time and effort for plasmid purification The kits are designed to efficiently isolate plasmid DNA from E coli in 1 5 2 5 hours using anion exchange columns without the use of any organic solvents or cesium chloride CsCl The isolated plasmid DNA purity is equivalent to two passes through CsCl gradients and has low endotoxin levels page 22 TM The PureLink HiPure Plasmid Filter Purification Kits are available in the following formats e The PureLink HiPure Plasmid Filter Midiprep and Maxiprep Kits allow you to purify plasmid DNA using different starting culture volumes page 7 e The PureLink HiPure Plasmid FP Filter and Precipitator Maxiprep Kit includes the PureLink HiPure Plasmid Filter Maxiprep Kit and PureLink HiPure Precipitator Module and allows plasmid DNA purification including isopropanol precipitation without centrifugation within one hour TM TM Continued on next page About the Kit Continued The HiPure T
28. ons on the label of the bottle Mix well Mark the bottle label to indicate that it contains RNase A 100 ug ml final concentration Store the buffer with RNase A at 4 C Lysis Buffer L7 Check the Lysis Buffer L7 for precipitates If present warm the solution briefly at 37 C to dissolve the precipitate Midiprep Procedure Introduction Before Starting Materials Needed Components Supplied with the Kit Note The PureLink HiPure Plasmid DNA Midiprep Kit allows purification of 100 350 ug of high quality plasmid DNA from 15 25 mL overnight E coli cultures in 2 hours when cloning high copy number plasmids Before beginning verify that the Resuspension Buffer R3 contains RNase A and no precipitate has formed in the Lysis Buffer L7 See page 9 for details e Overnight culture of transformed E coli cells page 7 e Isopropanol e 70 ethanol e Sterile microcentrifuge tubes e PureLink Nucleic Acid Purification Rack page 25 e Tubes or centrifuge bottles for harvesting cells e Centrifuge and rotor appropriate for harvesting cells e Appropriate 15 mL centrifuge tubes capable of withstanding centrifugation forces gt 12 000 x g e Centrifuge capable of centrifuging at gt 12 000 x g at 4 C e Optional PureLink HiPure Precipitator Module see page 25 e Resuspension Buffer R3 with RNase A e Lysis Buffer L7 e Precipitation Buffer N3 e Equilibration Buffer EQ1 e Wash Buffer W8 e Elu
29. ore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 2010 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Triton is a registered trademark of Union Carbide Chemicals amp Plastics Technology Corp invitrogen by Life technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
30. stituted Add 10 5 mL isopropanol to the elution tube containing the DNA see Eluting DNA page 16 Mix well Incubate for 2 minutes at room temperature Remove a 30 mL syringe supplied with the precipitator module from the package and remove the plunger Attach the PureLink HiPure Precipitator through the luer lock inlet to the 30 mL syringe nozzle Load the precipitated DNA mixture into the syringe place the precipitator over a waste container and insert the plunger into the syringe Use a slow constant force to push the plunger to pass the DNA mixture through the precipitator Discard the flow through Detach the precipitator from the syringe remove the plunger then reattach the precipitator to the syringe Note To prevent damage to the membrane do not remove the plunger while the precipitator is still attached to the syringe Continued on next page 18 Maxiprep Procedure Continued Precipitating DNA Using Precipitator Module Continued 19 10 11 12 13 14 T5 To wash the DNA precipitate Add 3 5 mL 70 ethanol into the syringe Place the precipitator over a waste container Insert the plunger into the syringe Push the plunger to pass the ethanol through the precipitator Detach the precipitator from the syringe remove the plunger then reattach the precipitator to the syringe To dry the precipitator membrane Insert the plunger into the syringe and push the plunger to pass air t
31. supernatant DNA sample into a fresh tube Store the purified DNA at 20 C or proceed to desired downstream application Note To avoid repeated freezing and thawing of DNA store the purified DNA at 4 C for immediate use or aliquot the DNA and store at 20 C for long term storage Continued on next page Maxiprep Procedure Continued N AMEND 7 oy NAN o ie E Precipitating DNA Using Precipitator Module Follow these guidelines when using the PureLink HiPure Precipitator Module see protocol below Always remove the precipitator module from the syringe before removing the plunger Do not apply excessive pressure while pushing the solution through the precipitator as too much pressure may detach the precipitator from the syringe Attach the precipitator to the syringe properly using the luer lock mechanism to avoid the detachment of the precipitator during sample processing Always use proper aseptic techniques when working with DNA and use only sterile DNase free tips and tubes to prevent DNase contamination When eluting the DNA with TE Buffer TE use a higher volume of TE Buffer to increase DNA yield Use alower volume of TE Buffer to increase DNA concentration see Elution Parameters in the precipitator module product insert The TE Buffer TE contains 10 mM Tris HCL pH 8 0 0 1mM EDTA If Tris HCl or EDTA interferes with downstream applications sterile water pH 8 0 can be sub
32. tion Buffer E4 e TE Buffer TE e HiPure Filter Midi Columns e Column Holder The protocol for the Midiprep kit of the PureLink HiPure Plasmid Filter Purification Kits is designed for purification of both high and low copy number plasmids without the need for adjusting buffer volumes Continued on next page 10 Midiprep Procedure Continued Equilibrating the Column Preparing Cell Lysate 11 TM The PureLink HiPure Filter Midi Columns are packaged with the Filtration Cartridge pre inserted into the column housing To Equilibrate the column 1 Use the Column Holder to support a HiPure Filter Midi Column in the mouth of a flask see page 9 or place the Midi Column on the PureLink Nucleic Acid Purification Rack see manual supplied with the rack for more details 2 Apply 15 mL Equilibration Buffer EQ1 directly to the filtration cartridge in the Midi Column 3 Allow the solution in the HiPure Filter Midi Column to drain by gravity flow Prepare the cell lysate see below while the HiPure Filter Midi Column is equilibrating 1 Harvest bacterial culture cells by centrifugation For high copy number plasmids harvest 15 25 mL of an overnight LB culture per sample in a 50 mL disposable tube For low copy number plasmids harvest 25 100 mL of an overnight LB culture per sample in a 50 mL disposable tube 2 Centrifuge the cells at 4 000 x g for 10 minutes to harvest the cells Remove all medium
33. ulture used of bacterial volumes or e Remove precipitated cell debris lysate from overgrown culture from overgrown cultures by HiPure Filter centrifuging the bacterial lysate Columns at 12000 x g for 5 minutes Inhibition of Presence of ethanol in Remove ethanol by air drying as downstream purified DNA described in the protocol reactions 24 Accessory Products Additional The following products are also available from Invitrogen Products For more details on these products visit our website at www invitrogen com or contact Technical Support page 26 Product Quantity Catalog no Quant iT DNA Assay Kit High Sensitivity 1000 assays Q33120 Quant iT DNA Assay Kit Broad Range 1000 assays Q33130 Qubit Fluorometer 1 each Q32857 PureLink Nucleic Acid Purification Rack 1 each K2100 13 PureLink HiPure Plasmid Miniprep 25 preps K2100 02 PureLink HiPure Plasmid Megaprep 4 preps K2100 08 PureLink HiPure Plasmid Gigaprep 2 preps K2100 09 PureLink HiPure BAC Buffer Kit 1 kit K2100 18 PureLink HiPure Precipitator Module 10 preps K2100 21 25 preps K2100 22 Luria Broth Base Miller s LB Broth Base 500 g 12795 027 powder 2 5 kg 12795 084 Ampicillin Sodium Salt irradiated 200 mg 11593 027 Carbenicillin Disodium Salt 5g 10177 012 E Gel E Gel Agarose Gels are bufferless pre cast agarose gels Agarose Gels with a variety of different agarose percentages and well and DNA formats designed for fast conv
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