NoEndo JETSTAR Endotoxin-Free Maxi/Mega/Giga Manual
Contents
1. 1 Harvest the cells by centrifuging the overnight LB culture at 12 000 x g for 3 minutes in a bucket Remove all medium Add 10 mL Cell Resuspending Buffer E1 with RNase A to the pellet and resuspend the cells until homogeneous Transfer cell suspension to a 50 mL centrifuge tube Note If overnight culture volume gt 200 mL use 20 mL Cell Resuspending Buffer E1 Add 10 mL Lysis Buffer E2 Mix gently by inverting the capped tube until the mixture is homogenous Do not vortex Incubate at room temperature for 5 minutes Note Do not allow lysis to proceed for more than 5 minutes If overnight culture volume gt 200 mL use 20 mL Lysis Buffer E2 Add 10 mL Precipitation Buffer E3 and mix immediately by inverting the capped tube until the mixture is homogeneous The viscous matter present after cell lysis step 3 should not remain Do not vortex Note When many samples are prepared in parallel mix each sample immediately after adding Precipitation Buffer E3 If overnight culture volume gt 200 mL use 20 mL Precipitation Buffer E3 Continued on next page Maxiprep Procedure Continued Preparing Cell Lysate Continued Removing Endotoxin Note 10 5 Centrifuge the mixture at gt 12 000 x g for 10 minutes at room temperature Note If the pellet does not adhere to the bottom of the tube incubate the tube at room temperature for 5 minutes to separate the lysate and pellet Pipette t2. Storing DNA To avoid repeated freezing and thawing of DNA store the purified DNA at 4 C for immediate use For long term storage aliquot the DNA and store at 20 C 20 Endotoxin free Gigaprep Experimental Overview Introduction Up to 10 mg of high quality plasmid DNA may be purified with the JetStar Endotoxin free Plasmid Gigaprep Kit in 2 3 hours using 2 5 L of high copy number starting bacterial culture or 5 L of low copy number starting bacterial culture Flow Chart The flow chart for purifying plasmid DNA using the JetStar 2 0 Endotoxin free Gigaprep Kit is shown below Remove Endotoxins Harvest E Coli Cells with Endo 1 Buffer Y Resuspend with Cell Load Sample onto Resuspending Buffer E1 DNA binding Cartridge y Lyse with Lysis Buffer E2 Wash with Endo 2 Buffer Y Precipitate with Precipitation Buffer E3 Wash with Wash Buffer E5 Gigaprep Using At AS Gigaprep Y D OR e f e JetFilter with Centrifugation Elute in Elution Buffer E6 Geck ECH Centrifuge for 20 minutes Wash JetFilter with Transfer to a Clean Wash Buffer E5 Container Wash and N ff Resuspend DNA d Precipitate DNA Ai Gigaprep Procedure Components Supplied with the Kit Note Materials Supplied by the User 22 Cell Resuspending Buffer E1 with RNase A page 23 Lysis Buffer E2 Precipitation Buffer E3 Endo 1 Buffer Equilibration Buffer E4 Endo 2 Buffer
3. system is compatible with other growth media including rich media we strongly recommend growing transformed E coli cells overnight in buffered LB Luria Bertani media for optimal results Use a bacterial culture in stationary phase The cell density should be approximately 1 x 10 cells per mL medium 1 1 5 Asoo units mL Continued on next page Before Starting Continued l DO 7 s O 0 LE NOT Creating an endotoxin free environment Note Follow the recommendations below to obtain the best results e Ensure that no DNase is introduced into the sterile buffers supplied with the kit e Sterilize all equipment coming in contact with DNA including pipette tips and tubes and maintain a sterile environment when handling DNA e Perform the recommended wash steps during purification to obtain the best results D To troubleshoot save aliquots as recommended during the purification procedure see page 31 e Bake all glassware for at least 6 hours at 200 C in an oven Autoclaving does NOT destroy endotoxins Since autoclaves are normally used to decontaminate bacterial cultures they are a common source of endotoxins e Use only plasticware that is certified to be endotoxin or pyrogen free by the respective vendor e If preparing buffers and solutions yourself all chemicals should be taken from fresh unopened bottles Reagents used to prepare endotoxin free buffers should be marked accordi
4. 2 0 Endotoxin free Plasmid Solution Low plasmid Temperature of Store Lysis Buffer E2 and Elution DNA yield Lysis Buffer E2 or Buffer E6 at room temperature Elution Buffer E6 is too low Low copy number Increase the volume of culture plasmid used processed Lysate at improper Carefully remove all culture medium pH or salt before resuspending cells concentration for Make sure that the correct volume of DNA binding Precipitation Buffer E3 is added JetStar DNA Make sure not to damage the cartridge Binding Cartridge during the procedure by screwing the damaged cartridge too tight or dropping it No plasmid Make sure to measure correctly the precipitation volume of eluate in each plasmid DNA centrifugation tube and add exactly present in eluate 0 7 volume of isopropanol aliquot but None or Make sure to centrifuge plasmid DNA little plasmid DNA at the appropriate speed and recovered after temperature in a swinging rotor precipitation Plasmid DNA pellet Do not dry the DNA pellet with a over dried vacuum system JetFilter JetFilter clogged by Let the lysate stand in the cartridge to clogged lysate allow the precipitate to float and form a layer on top of the lysate 32 Continued on next page Troubleshooting Continued Problem Cause Solution Genomic DNA Genomic DNA Gently invert tubes to mix after adding contamination sheared during Lysis Buffer E2
5. and Precipitation handling Buffer E3 Do not vortex as it can shear genomic DNA Additional Plasmid DNA Incubate lysate at room temperature plasmid forms permanently for no longer than 5 minutes after present denatured adding Lysis Buffer E2 RNA Lysate at improper Carefully remove all culture medium contamination pH salt before resuspending cells concentration or Make sure to add the correct volume temperature of Precipitation Buffer E3 to the lysate Lysate left on Once the lysate is loaded onto the JetStar DNA cartridge avoid delays in processing Binding Cartridge too long Lysate droplets remained on walls Wash droplets of lysate from walls of the cartridge with Wash Buffer E5 of cartridge RNase A digestion Make sure RNase A is added to Cell incomplete Resuspending Buffer E1 and Buffer E1 with RNase A has been properly stored at 4 C Use the recommended volume of Cell Resuspending Buffer E1 33 Accessory Products JetRack The JetRack Cat no 210 001 is designed for JetStar Maxi gravity flow columns It can be used simultaneously for 4 Maxi columns A waste container is included For more information visit www genomed dna com or contact Technical Support page 35 Additional The table below lists additional products available from Products Invitrogen that may be used with kits For more information about these products visit www invitrogen com or contact T
6. the cartridge so that no further liquid is pulled through the resin Let stand for 1 minute without agitation Apply vacuum again and draw the remaining liquid from the resin into the receiver bottle Keep vacuum on for approximately 5 minutes until all liquid has drained from the resin Optional Elution Add another 100 mL Elution Buffer E6 to the cartridge and repeat steps 2 4 Note This second elution step can increase the final DNA yield by approximately 10 The 250 ml flask contains eluate with the purified DNA Save a 100 uL aliquot of the eluate for further analysis Transfer the DNA eluate into several 50 mL centrifuge tubes maximum 17 5 mL per tube Add 0 7 volumes of isopropanol and mix well Note Proceed to the protocol described in the PureLink HiPure Precipitator manual after this step if you are using the precipitator Continued on next page Gigaprep Procedure Continued Eluting and 8 Centrifuge at gt 12 000 x g for 30 minutes at 4 C Precipitating Carefully remove and discard the supernatant DNA Note Perform centrifugations with a swinging bucket Continued rotor that allows gt 12 000 x g to prevent the plasmid DNA from sticking to the walls of the centrifuge tube If such a rotor is not available siliconize the centrifuge tubes with a repellent silane dimethyldichlorosilane 9 Wash the DNA pellet in 10 15 mL endotoxin free 70 ethanol Vortex gently 10 Centrifuge at gt 12 000
7. Kit Materials Supplied by the User Cell Resuspending Buffer E1 with RNase A see page 8 Lysis Buffer E2 Precipitation Buffer E3 Endo 1 Buffer Equilibration Buffer E4 Endo 2 Buffer Wash Buffer E5 Elution Buffer E6 TE Buffer Endotoxin free water for preparing 70 ethanol JetStar Maxi Columns Overnight culture of transformed E coli cells Isopropanol Sterile endotoxin or pyrogen free microcentrifuge tubes JetRack page 34 Tubes or buckets of appropriate size for harvesting cells Centrifuge and rotor appropriate for harvesting cells Appropriate 50 mL centrifuge tube capable of withstanding centrifugation forces gt 12 000 x g Centrifuge capable of centrifuging at gt 12 000 x g at 4 C Optional PureLink HiPure Precipitator Module page 34 Continued on next page Maxiprep Procedure Continued Purification Rack Overnight Culture Preparing Buffers and Solutions The JetRack is designed for use with the JetStar Plasmid DNA Maxiprep Kits see page 34 for ordering information The JetRack consists of a Column Holder Rack for processing 4 maxiprep columns simultaneously a Collection Tube Rack and a Waste Container for collecting waste Inoculate a plasmid containing E coli culture according to good microbiological practice in growth medium with the appropriate antibiotic and grow the cells for 16 18 hours usually overnight or 24 36 hours if using a buf
8. M Sodium acetate pH 5 0 0 6 M NaCl 0 15 v v Triton X 100 Endo 2 Buffer Proprietary formulation Wash Buffer E5 0 1 M Sodium acetate pH 5 0 0 8 M NaCl Elution Buffer E6 0 1 M Tris HCI pH 8 5 1 25 M NaCl TE Buffer 10 mM Tris HCl pH 8 0 8 5 0 1 mM EDTA viii Introduction About the Kits The JetStar JetStar Maxi Mega and Giga Endotoxin free Plasmid Tech nology Purification Kits contain a unique anion exchange resin packed in a gravity flow column maxiprep or a vacuum driven filter cartridge mega and gigaprep JetStar Endotoxin free Plasmid Kits are the ideal tool to efficiently isolate large quantities of ultrapure plasmid DNA with an endotoxin content of lt 0 001 endotoxin units ug EU yg Advantages Use of the JetStar Endotoxin free Plasmid Purification Kits to isolate plasmid DNA provides the following advantages Rapid and convenient purification method that allows high yield plasmid purification in 2 3 hours Unique anion exchange resin technology to isolate plasmid DNA negates the need for any organic solvents cesium chloride CsCl or vacuum manifolds Endotoxin content of purified DNA lt 0 001 EU pg Maxiprep utilizes fast gravity flow columns and mega and gigaprep utilize ultra quick vacuum cartridges No additional incubation times compared to the standard JetStar Plasmid Purification protocol Bacterial lysate remains clear during endotoxin removal Buffers
9. 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 Continued on next page Purchaser Notification Continued Limited Use Label License No 5 Invitrogen Technology The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a
10. the precipitate to float and form a layer on top of the lysate If necessary extend the incubation time of the lysate in the JetFilter until the layer has formed to prevent clogging and ensure efficient filtration c Connect the vacuum source to the tubing connector of the JetFilter and apply vacuum 600 to 800 mbar until all liquid has drained Turn off the vacuum Note An unclogged JetFilter filters 125 mL of filtrate in 1 2 minutes when vacuum is applied Continued on next page 26 Gigaprep Procedure Continued Preparing Cell d Add 50 mL of Wash Buffer E5 to the JetFilter and Lysate gently stir the precipitate with a sterile spatula to Continued improve the flow of liquid through the filter unit e Connect the vacuum source again and apply vacuum until all liquid has been pulled through completely Save a 100 uL aliquot of the flow through for further analysis f Close the bottle and mix the contents well by shaking Gentle agitation of the precipitate improves the flow of liquid through the filter unit The bottle now contains the clear filtered bacterial lysate with the plasmid DNA When filtration is finished discard the single use JetFilter g Proceed to Removing Endotoxin below Removing 1 To the supernatant add 1 10 volume of Endo 1 Buffer and Endotoxin mix well to homogeneity For example add 35 mL of Endo 1 Buffer to 350 mL cleared lysate Mix well by inverting 2 Load the supernatan
11. x g for 10 minutes at 4 C Carefully remove and discard the supernatant 11 Air dry the pellet for 10 15 minutes at room temperature Note Be careful when drying the DNA pellet under vacuum in a speed vac or in a vacuum chamber The DNA will not redissolve completely if it is overdried 12 Resuspend the DNA pellet in a suitable volume of endotoxin free TE Buffer supplied with the kit or endotoxin free 10 mM Tris HCl or endotoxin free water Storing DNA 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 29 Appendix Estimating DNA Yield Introduction DNA Yield 30 Once you have isolated DNA you may determine the quantity of the purified DNA as described below Perform DNA quantitation using UV absorbance at 260 nm or Quant iT DNA Assay Kits see page 34 for ordering information UV Absorbance 1 Prepare a dilution of the DNA solution in 10 mM Tris HCl pH 7 5 Mix well Measure the absorbance of the dilution at 260 nm A260 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 ng mL A260 x 50 x dilution factor For DNA A260 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 iI DNA
12. 0 Endotoxin free Megaprep Kits is shown below Remove Endotoxins Harvest E Coli Cells p with Endo 1 Buffer L Resuspend with Cell Load Sample onto Resuspending Buffer E1 DNA binding Cartridge Y Lyse with Lysis Buffer E2 Wash with Endo 2 Buffer Y Precipitate with Precipitation Buffer E3 Wash with Wash Buffer E5 Megaprep Using we a Megaprep i OR i JetFilter with Centrifugation Elute in Elution Buffer E6 eau ale Centrifuge for 20 minutes SS REES p Precipitate DNA Wash JetFilter with Transfer to a Clean Wash Buffer E5 Container H Wash and Si SI Resuspend DNA Megaprep Procedure Components Supplied with the Kit Note Materials Supplied by the User Cell Resuspending Buffer E1 with RNase A page 15 Lysis Buffer E2 Precipitation Buffer E3 Endo 1 Buffer Equilibration Buffer E4 Endo 2 Buffer Wash Buffer E5 Elution Buffer E6 TE Buffer Endotoxin free water for preparing 70 ethanol Megaprep JetFilter Cat no 233006 only JetStar DNA Binding Cartridge The JetFilter for clarification of the bacterial lysate contains a yellow ring JetStar DNA Binding Cartridges with endotoxin free ion exchange chromatography material for purifying DNA contain a red ring Overnight culture of transformed E coli cells Isopropanol Sterile endotoxin or pyrogen free microcentrifuge tubes Tubes or buckets of appropriate size for harvesting cells Centrifug
13. 6 preps 233006 JetStar 2 0 Endotoxin free Gigaprep Kit 6 preps 242006 JetStar 2 0 Endotoxin free Gigaprep JetFilter Kit 6 preps 243006 Intended Use For research use only Not intended for human or animal diagnostic or therapeutic uses Continued on next page Kit Contents and Storage Continued JetStar 2 0 Endotoxin free Maxiprep Kits vi TM The components included in the JetStar 2 0 Endotoxin free Maxiprep Kits are listed below Store RNase A at 4 C and store all other components at room temperature See page viii for buffer compositions Item Cat no Cat no Cat no 222010 222020 222100 10 preps 20 preps 100 preps Cell Resuspending Buffer E1 110 mL 220 mL 2 x 550 mL RNase A 20 mg mL 650 pL 1 5 mL 2x3mL Lysis Buffer E2 110 mL 220 mL 2 x 550 mL Precipitation Buffer E3 110 mL 220 mL 2 x 550 mL Endo 1 Buffer 33 mL 66 mL 330 mL Equilibration Buffer E4 330 mL 660 mL 2 x 1 650 mL Endo 2 Buffer 330 mL 660 mL 2 x 1 650 mL Wash Buffer E5 330 mL 660 mL 2x 1 650 mL Elution Buffer E6 165 mL 330 mL 1 650 mL Endotoxin free Water 30 mL 30 mL 170 mL TE Buffer 30 mL 50 mL 200 mL Maxi Columns 10 each 20 each 100 each Continued on next page Kit Contents and Storage Continued JetStar 2 0 TM The components included in the JetStar 2 0 Endotoxin free Endotoxin Mega and G
14. 9 0 5732 904700 Fax 49 0 5732 9047010 E mail techservice genomed dna com 35 Purchaser Notification Limited Warranty 36 GENOMED 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 GENOMED 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 GENOMED makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore 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
15. Assay Kits page 34 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 fluorometer or Qubit Fluorometer Troubleshooting 7 If problems arise during the procedure analyze the aliquots harvested during the procedure on a 1 agarose gel to E determine the presence of DNA in the aliquots The table S below lists the results and probable cause Aliquot Normal Abnormal Cause of Abnormal Results Results Results Lysate at improper pH or salt JetStar DNA concentration to bind column Binding JetStar DNA Binding Cartridge flow No DNA DNA Cartridge overloaded DEE JetStar DNA Binding Cartridge damaged JetStar DNA Si SC Binding NoDNA DNA ee SE Cartridge Wash SE SE Lysate at improper pH or salt JetStar DNA concentration to bind column Binding DNA No DNA Use correct Elution Buffer E6 Cartridge Eluate and perform optional elution step Continued on next page 31 Troubleshooting Continued Introduction Problem Review the information below to troubleshoot your experiments with JetStar Purification Kits Cause TM
16. Le Genomed JetStar Endotoxin free Plasmid Purification Kits For purifying transfection grade plasmid DNA Cat nos 222010 222020 222100 232006 233006 242006 and 243006 Rev Date 20 April 2010 Manual part no 70 15015 MAN 0001741 User Manual Table of Contents Kit Contents and Storage sisi see ge EES EEN v e E EE 1 About TEE 1 E de 4 Before taggen 4 Endotoxin free MaxiPprepP sosesseoseossosesssossssssonersensrrsrnsrsrrsersrrnnrsnnnrn nns 6 Exp rimental Cherrier dee 6 Maxiprep Procedure eebe EES 7 Endotoxin free Megaprep ccssssssssssssssssssscecscsssssssssssesesssssseseseseees 12 Experimental Overview REENEN 12 Me gaprep Procedures geesde 13 Endotoxin free Gigaprep esssseesessessesroreesesseseeseosceresseseesesneosessoseesessesee 21 Experimental OVervie Waipiro eani iain a i a SiR U EAR ents 21 EE e ne N E EA INE 22 APPENCIK E A E eege 30 Estimating DNA E 30 Troubleshooting senses a EES 31 Accessory ProdUCiS issen tinse eirinn e R a iea 34 Technical Support isesi ien ie rae apai S i a ai huts 35 EurchaserNoptteation deiere 36 Kit Contents and Storage Types of Kits This manual is supplied with the following products Product Quantity Cat no JetStar 2 0 Endotoxin free Maxiprep Kit 10 preps 222010 20 preps 222020 100 preps 222100 JetStar 2 0 Endotoxin free Megaprep Kit 6 preps 232006 JetStar 2 0 Endotoxin free Megaprep JetFilter Kit
17. Wash Buffer E5 Elution Buffer E6 TE Buffer Endotoxin free water for preparing 70 ethanol Gigaprep JetFilter for Cat no 243006 only JetStar Gigaprep DNA Binding Cartridge JetFilters for clarification of the bacterial lysate contain a yellow ring JetStar Gigaprep DNA Binding Cartridges with endotoxin free ion exchange chromatography material for purifying DNA contain a red ring Overnight culture of transformed E coli cells Isopropanol Sterile endotoxin or pyrogen free microcentrifuge tubes Tubes or buckets of appropriate size for harvesting cells Centrifuge and rotor appropriate for harvesting cells Sterile 50 ml centrifuge tubes Sterile spatula Sterile container for transferring the lysate Continued on next page Gigaprep Procedure Continued Materials e Vacuum source capable of generating a negative Supplied by pressure of 600 to 800 mbar and equipped with a the User vacuum regulator Continued Note If the vacuum pump is not equipped with a vacuum regulator you may need to purchase a regulator such as the PureLink vacuum regulator see page 34 and attach the regulator to the vacuum pump e 1L flask or a 1 000 mL Stericup receiver flask with 45 mm thread Millipore Cat no SCOOB10RE Note You will need two 1 Liter flasks for each sample if you are using a JetFilter for lysate clarification e Sterile endotoxin free 250 mL flask with 45 mm thread e Optional PureLink HiP
18. c may crack 2 Connect the vacuum source to the tubing connector of the filtration cartridge An example of the set up with a JetStar DNA Binding Cartridge is shown in the figure on the next page Note Make sure the vacuum source has a vacuum regulator to adjust the vacuum pressure during the procedure see page 34 3 Equilibrate the JetStar DNA Binding Cartridge with 200 mL Equilibration Buffer E4 4 Apply vacuum to the cartridge through the side arm with tubing connector Keep vacuum on until all liquid has drained from the resin Discard the flow through Set equilibrated cartridge aside for later use Removing Endotoxin page 27 Continued on next page 24 Gigaprep Procedure Continued JetStar DNA Binding Cartridge Preparing the JetFilter Preparing Cell Lysate DNA binding cartridge Line to Vacuum vacuum Regulator source Attach a JetFilter with a yellow ring onto a clean 1 000 mL bottle with a 45 mm neck Do not overtighten the cartridge on the bottle neck since the cartridge plastic may crack Set cartridge aside for later use see Step 5 page 26 Harvest up to 2 5 L high copy number plasmids or up to 5 L low copy number plasmids of E coli cells from overnight LB cultures by centrifuging cells in a bucket at 12 000 x e for 5 minutes at 4 C Remove all medium Add 125 mL Cell Resuspending Buffer E1 with RNase A to the pellet and resuspend the cells
19. crucial for endotoxin removal are certified to contain lt 0 1 EU mL No chromosomal DNA contamination Ability to purify all types and sizes of plasmid DNA Reliable performance of the purified plasmid DNA ina variety of downstream applications Continued on next page About the Kits Continued Procedure Overview The JetStar Maxi Mega and Giga Endotoxin free Plasmid Purification Kits use a patented anion exchange resin to purify plasmid DNA The patented resin provides excellent capacity with fast flow rates high resolution high yield and efficient endotoxin removal The isolated plasmid DNA has a purity equivalent to two passes through CsCl gradients The general procedure is carried out as follows 1 E coli cells are harvested resuspended in Cell Resuspending Buffer E1 with RNase and then lysed with Lysis Buffer E2 Precipitation Buffer E3 is added to the lysate and the lysate is clarified by centrifugation Maxi Mega and Gigapreps using centrifugation or by passing over a vacuum assisted pre packed lysate binding filter Mega and Gigaprep Using JetFilters Endotoxin is preliminarily removed with Endo 1 Buffer The cleared lysate is then passed through a pre packed anion exchange column The negatively charged phosphates on the backbone of the DNA interact with the positive charges on the surface of the resin The temperature salt concentration and pH of the solutions influence binding Under
20. e and rotor appropriate for harvesting cells Sterile 50 mL centrifuge tubes Sterile spatula Sterile container for transferring the lysate Continued on next page 13 Megaprep Procedure Continued Materials e Vacuum source capable of generating a negative Supplied by pressure of 600 to 800 mbar and equipped with a the User vacuum regulator Continued Note If the vacuum pump is not equipped with a vacuum regulator you may need to purchase a regulator such as the PureLink vacuum regulator see page 34 and attach the regulator to the vacuum pump e 1L flask or a 1 000 mL Stericup Receiver flask with 45 mm thread Millipore Cat no SCOOB10RE e Sterile endotoxin free 250 mL flask with 45 mm thread e For plasmid purification using the JetFilter Cat no 233006 you will also need a 500 mL flask or a 500 mL Stericup Receiver Flask with 45 mm thread Millipore Cat no SCOOBO5RE e Optional PureLink HiPure Precipitator Module page 34 Overnight Inoculate a plasmid containing E coli culture according to Culture good microbiological practice in growth medium with the appropriate antibiotic and grow the cells for 16 18 hours or for 24 36 hours if using a buffered growth medium e For high copy number plasmids use 500 mL of an overnight LB culture per sample e For low copy number plasmids use 2 500 mL of an overnight LB culture per sample Continued on next page 14 Megaprep Procedu
21. echnical Support page 35 Product Amount Cat no Luria Broth Base Miller s LB Broth Base powder 500 g 12795 027 2 5 kg 12795 084 Ampicillin Sodium Salt irradiated 200 mg 11593 027 Carbenicillin Disodium Salt 5g 10177 012 PureLink Vacuum Regulator 1 each K2110 02 PureLink HiPure Precipitator Module 10 preps K2100 21 25 preps K2100 22 Quant iT DNA Assay Kit High Sensitivity 1 000 assays Q33120 Quant iT DNA Assay Kit Broad Range 1 000 assays Q33130 Qubit Fluorometer 1 each Q32857 E Gel E Gel Agarose Gels are bufferless pre cast agarose gels Agarose Gels designed for fast convenient electrophoresis of DNA samples and DNA E Gel agarose gels are available in different agarose Ladders percentages and well formats for your convenience DNA ladders are available from Invitrogen for sizing DNA For more details on these products visit www invitrogen com 34 Technical Support World Wide Visit the website at www genomed dna com for Web e Technical resources including manuals MSDSs FAQs e Complete technical support contact information e Access to the 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 www genomed dna com GENOMED GmbH Poststr 22 D 32584 L hne Germany Tel 4
22. edure Continued Removing Endotoxin Continued Note Eluting and Precipitating DNA 5 Repeat step 4 using 150 mL Wash Buffer E5 Discard the flow through The binding cartridge contains the DNA 6 Proceed to Eluting and Precipitating DNA below TM The PureLink HiPure Precipitator Module page 34 may be used for precipitation in 10 minutes without any centrifugation steps Alternatively follow the protocol on the nest page to perform traditional DNA precipitation using centrifugation 1 Remove the JetStar DNA Binding Cartridge from the waste bottle and attach it onto a clean sterile endotoxin free 250 mL Stericup Receiver Flask Add 50 mL Elution Buffer E6 into the cartridge 3 Apply soft vacuum 100 to 200 mbar to the cartridge through the side arm until approximately 5 10 mL of Elution Buffer E6 has passed through the cartridge Then release the vacuum from the cartridge so that no further liquid is pulled through the resin 4 Let stand for 1 minute without agitation 5 Apply vacuum again and draw the remaining liquid from the resin into the receiver bottle Keep vacuum on for approximately 5 minutes until all liquid has drained from the resin 6 Optional Elution Add an additional 50 mL Elution Buffer E6 to the JetStar DNA Binding Cartridge Repeat Steps 3 5 Note This second elution step can increase the final DNA yield by approximately 10 15 The 250 mL receiver flas
23. fered growth medium e For high copy number plasmids use 100 mL of an overnight LB culture per sample e For low copy number plasmids use 250 500 mL of an overnight LB culture per sample Note For culture volumes gt 200 mL add twice the amount of Cell Resuspending Buffer E1 with RNase A Lysis Buffer E2 and Precipitation Buffer E3 as directed in Steps 2 3 and 4 next page Cell Resuspending Buffer E1 Add RNase A to the Cell Resuspending Buffer E1 according to the note with the RNase A tube Mix well The final RNase A concentration is 100 pg mL Mark the bottle label to indicate that RNase A is added and store at 4 C Lysis Buffer E2 Check the Lysis Buffer E2 for precipitates If present warm the solution briefly at 37 C to dissolve the precipitate 70 Ethanol Prepare 70 ethanol with the endotoxin free water supplied with the kit Add absolute 96 100 ethanol as stated on the label of the bottle and mix thoroughly Continued on next page Maxiprep Procedure Continued Equilibrating the Column Preparing Cell Lysate TM Place the JetStar Maxi column on the JetRack see the manual supplied with the rack for more details Apply 30 mL Equilibration Buffer E4 to the column Allow the solution in the column to drain by gravity flow Equilibration takes about 10 15 minutes but does not influence the speed of the protocol therefore proceed to Step 1 below during equilibration
24. fetech com 2010 Life Technologies Corporation All rights reserved 37 Corporate Headquarters Genomed GmbH Poststr 22 D 32584 Lohne Germany Tel 49 0 5732 904700 Fax 49 0 5732 9047010 techservice genomed dna com www genomed dna com
25. he clear lysate into another sterile tube and centrifuge at gt 12 000 x g for 5 minutes at room temperature to remove any remaining cellular debris Proceed to Removing Endotoxin below Mix the clear supernatant with 1 10 volume of Endo 1 Buffer For example add 3 mL of Endo 1 Buffer to 30 mL cleared lysate Mix well by inverting Note Mix the cleared lysate and Endo 1 Buffer to homogeneity Load the supernatant onto the equilibrated column with a pipette Allow the solution in the column to drain by gravity flow Wash the column with 30 mL Endo 2 Buffer Allow the solution in the column to drain by gravity flow Discard the flow through Wash the column with 30 mL Wash Buffer E5 as described above Proceed to Eluting and Precipitating DNA next page The PureLink HiPure Precipitator Module page 34 may be used for precipitation in 10 minutes without any centrifugation steps Alternatively follow the protocol on the next page to perform traditional DNA precipitation using centrifugation Continued on next page Maxiprep Procedure Continued Eluting and 1 Place a sterile endotoxin or pyrogen free 30 mL Precipitating centrifuge tube elution tube under the column DNA 2 Add 15 mL Elution Buffer E6 to the 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 column 3 Add 10 5 mL i
26. iga prep Kits are listed below Note that the kits free Mega are available with and without the JetFilter Store RNase A and Giga Kits at 4 C and all other components at room temperature Item Cat no Cat no Cat no Cat no 232006 233006 242006 243006 6 preps 6 preps 6 preps 6 preps Cell Resuspending Buffer E1 330 mL 330 mL 825 mL 825 mL RNase A 20 mg mL 3 mL 3 mL 2x3mL 2x3mL Lysis Buffer E2 330 mL 330 mL 825 mL 825 mL Precipitation Buffer E3 330 mL 330 mL 825 mL 825 mL Endo 1 Buffer 99 mL 99 mL 250 mL 250 mL Equilibration Buffer E4 660 mL 660 mL 2x 660 mL 2 x 660 mL Endo 2 Buffer 990 mL 990 mL 1 980 mL 1 980 mL Wash Buffer E5 990 mL 990 mL 1 980 mL 1 980 mL Elution Buffer E6 330 mL 330 mL 660 mL 660 mL Endotoxin free Water 30 mL 30 mL 30 mL 30 mL TE Buffer 50 mL 50 mL 200 mL 200 mL Lysate Filtration JetFilter none 6 each none 6 each DNA binding Cartridge 6each 6each 6 each 6 each Continued on next page vii Kit Contents and Storage Continued Buffer The composition of buffers included in the JetStar Composition Endotoxin free Kits is listed below Buffer Composition Cell Resuspending Buffer E1 50 mM Tris HCl pH 8 0 10 mM EDTA RNase A 20 mg mL in Buffer E1 Lysis Buffer E2 0 2 M NaOH 1 w v SDS Precipitation Buffer E3 3 1 M Potassium acetate pH5 5 Endo 1 Buffer Proprietary formulation Equilibration Buffer E4 0 1
27. k now contains the eluate with the purified DNA Save a 100 uL aliquot of the eluate for further analysis Continued on next page 19 Megaprep Procedure Continued Eluting and 7 Transfer the DNA eluate into several 50 mL centrifuge Precipitating tubes maximum 17 5 mL per tube DNA 8 Add 0 7 volumes of isopropanol and mix well Continued Note Proceed to the protocol described in the PureLink HiPure Precipitator manual after this step if you are using the precipitator 9 Centrifuge at gt 12 000 x g for 30 minutes at 4 C Carefully remove and discard the supernatant Note Perform centrifugations with a swinging bucket rotor that allows gt 12 000 x g to prevent the plasmid DNA from sticking to the walls of the centrifuge tube If such a rotor is not available siliconize the centrifuge tubes with a repellent silane dimethyldichlorosilane 10 Wash the DNA pellet in 10 15 mL endotoxin free 70 ethanol Vortex gently 11 Centrifuge at gt 12 000 x g for 10 minutes at 4 C Carefully remove and discard the supernatant 12 Air dry the pellet for 10 15 minutes at room temperature or 65 70 C Note Be careful when drying the DNA pellet under vacuum in a speed vac or in a vacuum chamber The DNA will not be redissolved completely if it is overdried 13 Resuspend the DNA pellet in a suitable volume of endotoxin free TE Buffer supplied with the kit or endotoxin free 10mM Tris HCl or endotoxin free water
28. moderate salt conditions plasmid DNA remains bound to the resin while residual endotoxin is washed away with Endo 2 Buffer RNA proteins carbohydrates and other impurities are then washed away with Wash Buffer E5 The plasmid DNA is eluted under high salt conditions with the Elution Buffer E6 The eluted DNA is desalted and concentrated with an alcohol precipitation step The entire protocol can be completed in 2 3 hours Continued on next page About the Kits Continued System Specifications e May be used for all sizes of plasmid DNA e Ar60 Argo gt 1 85 JetStar Endotoxin free Plasmid Purification Kits have the following specifications Specification Maxiprep Megaprep Gigaprep Starting Culture Volume 100 200 mL 500 mL 2 5L Column Procedure Time 45 minutes 10 minutes 10 minutes JetFilter Reservoir Capacity n a 230 mL 450 mL ee Cartridge I 60 mL 200 mL 400 mL Elution Volume 15 mL 50 mL 100 mL DNA Yield up to 750 pg upto2 5mg upto 10 mg DNA Recovery 90 95 90 95 90 95 Downstream DNA purified using JetStar Plasmid Purification Kits is at a Applications purity equivalent to two passes through CsCl gradients and is suitable for use in a variety of downstream applications including those requiring the highest purity such as e Transfection of primary cells including neuronal cells e Microinjection into animals and eukaryotic cells e Gene therap
29. ngly to keep the chemicals endotoxin free When working with gram negative bacteria in the lab endotoxins are likely to be present on nearly all parts that are in use by multiple researchers e g many parts of glassware Endotoxin molecules are very resistant against physical means of inactivation e g heat Therefore consider even single use items e g plasticware disposables to be contaminated with endotoxins if not expressively otherwise stated Following elution be very careful not to re contaminate plasmid DNA again by using endotoxin contaminated water chemicals glass or plasticware Endotoxin free Maxiprep Experimental Overview Introduction Up to 750 ug of high quality plasmid DNA may be purified Kit in with the JetStar Endotoxin free Plasmid Maxiprep 2 hours from overnight E coli cultures JetStar 2 0 The flow chart for purifying plasmid DNA using the Endotoxin JetStar 2 0 Endotoxin free Maxiprep Kit is shown below free Maxiprep Kit MaxiPrep 30 mL Equilibration Load Buffer E4 Column I Harvest Cells 30 mL Endo 2 Buffer gt Sim Y V 10 mL Cell Resuspending 30 mL Wash Buffer E1 Buffer E5 mm l aA 10 mL Lysis e Buffer E2 15 mL Elution g Buffer E6 y 10 mL Precipitation Y d Buffer E3 10 5 mL gt lsopropanol D Y 5mL 1 10 volume 70 Ethanol Endo 1 Buffer W TE Buffer Maxiprep Procedure Components Supplied with the
30. party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 or outlicensing li
31. rce to the tubing connector of the filtration cartridge and apply vacuum 600 to 800 mbar Collect the flow through into the flask Keep the vacuum on until all liquid has drained Turn off vacuum Note An unclogged JetFilter filters 125 mL of filtrate in 1 2 minutes when vacuum is applied Add 50 mL of Wash Buffer E5 to the JetFilter and gently stir the precipitate with a sterile spatula to improve the flow of liquid through the filter unit Apply vacuum until all liquid has drained from the filtration cartridge The receiving flask contains the clarified lysate with the plasmid DNA Remove and discard the JetFilter into biohazardous waste Pour the contents of the receiving flask filtered lysate into a new sterile container Proceed to Removing Endotoxin below Mix the supernatant with 1 10 volume of Endo 1 Buffer For example add 15 mL of Endo 1 Buffer to 150 mL cleared lysate Mix well by inverting until the mixture is homogenous Load the supernatant onto the equilibrated JetStar DNA Binding Cartridge with a red ring from Step 4 page 16 Apply vacuum to the cartridge Keep the vacuum on until all liquid has drained from the resin Take an aliquot 100 uL of the flow through for further analysis Add 150 mL of Endo 2 Buffer to the cartridge and apply vacuum Keep the vacuum on until all liquid has drained from the resin Take a 100 uL aliquot of the flow through Continued on next page Megaprep Proc
32. re Continued Preparing Cell Resuspending Buffer E1 Buffers and Add RNase A to the Cell Resuspending Buffer E1 according to Solutions the note with the RNase A tube Mix well The final RNase A concentration is 100 pg mL Mark the bottle label to indicate that RNase A is added Store Cell Resuspending Buffer E1 with RNase A at 4 C Lysis Buffer E2 Check the Lysis Buffer E2 for precipitates If present warm the solution briefly at 37 C to dissolve the precipitate 70 Ethanol Prepare 70 ethanol with the endotoxin free water supplied with the kit Add the appropriate amount of absolute 96 100 ethanol as stated on the label of the bottle and mix thoroughly Preparing the 1 Attach a JetFilter with a yellow ring onto a clean JetFilter 500 mL bottle with a 45 mm neck Do not overtighten the cartridge on the bottle neck since the cartridge plastic may crack 2 Set cartridge aside for later use Clarifying the Lysate Using the JetFilter page 17 Preparing the 1 Attach a JetStar Megaprep DNA Binding Cartridge JetStar DNA with a red ring onto a clean 1 000 mL Stericup receiver Binding flask or a 1 Liter bottle with 45 mm neck Do not Cartridge overtighten the cartridge on the bottle neck since the cartridge plastic may crack 2 Connect the vacuum source to the tubing connector of filtration cartridge An example of the set up with a JetStar Megaprep DNA Binding Cartridge is shown in the figu
33. re on the next page Note Make sure the vacuum source has a vacuum regulator to adjust the vacuum pressure during the procedure see page 34 Continued on next page 15 Megaprep Procedure Continued Preparing the JetStar DNA Binding Cartridge Continued Preparing Cell Lysate 16 3 Equilibrate the JetStar DNA Binding Cartridge with 100 mL Equilibration Buffer E4 Apply vacuum to the cartridge through the side arm with tubing connector Keep vacuum on until all liquid has drained from the resin Discard the flow through Set equilibrated cartridge aside for later use Removing Endotoxin page 18 DNA binding cartridge Pi A Ss A Line to Vacuum vacuum Regulato source we 1 Harvest up to 500 mL high copy number plasmid or up to 2 5 L low copy number plasmid of E coli cells by centrifuging the overnight LB culture at 12 000 x g for 5 minutes in a bucket Remove all medium Add 50 mL Cell Resuspending Buffer E1 with RNase A to the pellet and resuspend the cells until homogeneous Continued on next page Megaprep Procedure Continued Preparing Cell Lysate Continued Clarifying the Lysate Using the JetFilter 3 Add 50 mL Lysis Buffer E2 Mix gently by inverting the capped tube until the mixture is homogenous Do not vortex Incubate at room temperature for 5 minutes Note Do not allow lysis to proceed for more than 5 minutes Add 50 mL P
34. recipitation Buffer E3 and mix immediately by inverting the capped tube until the mixture is homogeneous The viscous matter present after cell lysis Step 3 should not remain Do not vortex Note When many samples are prepared in parallel each sample should be mixed immediately after adding Lysis Buffer E3 The cell lysate may be clarified using centrifugation or vacuum e For Cat no 232006 clarify the lysate by centrifuging the lysate at gt 12 000 x g for 20 minutes at room temperature and transfer the particle free supernatant into a clean container Proceed directly to Removing Endotoxin page 18 e For Cat no 233006 clarify the lysate using the JetFilter as directed in Clarifying the Lysate Using the JetFilter below 1 Pour the neutralized bacterial lysate directly into the prepared JetFilter with the yellow ring see Preparing the JetFilter page 15 Let stand at room temperature for at least 10 minutes without agitation Note Let the lysate stand for at least 10 minutes after the transfer into the cartridge to allow the precipitate to float and form a layer on top of the lysate If necessary extend the incubation time of the lysate in the cartridge until the layer has formed to prevent clogging and ensure efficient filtration Continued on next page 17 Megaprep Procedure Continued Clarifying the Lysate Using the JetFilter Continued Removing Endotoxin 18 Connect the vacuum sou
35. sopropanol to the elution tube Mix well Note Proceed to the protocol described in the PureLink HiPure Precipitator manual after this step if you are using the precipitator 4 Centrifuge at gt 12 000 x g for 30 minutes at 4 C Carefully remove and discard the supernatant 5 Wash the DNA pellet in 5 mL endotoxin free 70 ethanol 6 Centrifuge at gt 12 000 x g for 5 10 minutes at 4 C Carefully remove and discard the supernatant 7 Air dry the pellet for 10 15 minutes at room temperature or 65 70 C Note Be careful when drying the DNA pellet under vacuum in a speed vac or in a vacuum chamber The DNA will not redissolve completely if it is overdried 8 Resuspend the DNA pellet in a suitable volume of endotoxin free TE Buffer supplied with the kit or endotoxin free 10 mM Tris HCl or endotoxin free water Storing DNA To avoid repeated freezing and thawing of DNA store the purified DNA at 4 C for immediate use For long term storage aliquot the DNA and store at 20 C 11 Endotoxin free Megaprep Experimental Overview Introduction Up to 2 5 mg of high quality plasmid DNA may be purified with the JetStar Endotoxin free Plasmid Megaprep Kit The JetStar Megaprep procedure is completed in 2 3 hours using 500 mL of high copy number starting bacterial culture or 2 5 L of low copy number starting bacterial culture Flow Chart The flow chart for purifying plasmid DNA using the JetStar 2
36. t onto the equilibrated JetStar DNA Binding Cartridge with a red ring from Step 4 page 24 Apply vacuum to the cartridge through the side arm with tubing connector Keep the vacuum on until all liquid has drained from the resin Take an aliquot 100 uL of the flow through for further analysis if desired 3 Add 275 mL of Endo 2 Buffer to the JetStar DNA Binding Cartridge and apply vacuum Keep the vacuum on until all liquid has drained from the resin Take a 100 uL aliquot of the flow through for further analysis if desired 4 Repeat Step 3 using 275 mL Wash Buffer E5 Discard the flow through The binding cartridge contains the DNA 5 Proceed to Eluting and Precipitating DNA next page Continued on next page 27 Gigaprep Procedure Continued Note Eluting and Precipitating DNA 28 TM The PureLink HiPure Precipitator Module page 34 may be used for precipitation in 10 minutes without any centrifugation steps Alternatively follow the protocol below to perform traditional DNA precipitation using centrifugation Remove the JetStar DNA Binding Cartridge from the waste bottle and attach it onto a clean sterile endotoxin free 250 mL Stericup receiver flask or equivalent Add 100 mL Elution Buffer E6 into the cartridge Apply a soft vacuum 100 to 200 mbar to the cartridge until approximately 20 30 mL of Elution Buffer E6 has eluted from the cartridge Then release the vacuum from
37. until homogeneous Add 125 mL Lysis Buffer E2 Mix gently by inverting the capped tube until the mixture is homogenous Do not vortex Incubate at room temperature for 5 minutes Note Do not allow lysis to proceed for more than 5 minutes Continued on next page 25 Gigaprep Procedure Continued Preparing Cell 4 Add 125 mL Precipitation Buffer E3 and mix immediately Lysate by inverting the capped tube until the mixture is Continued homogeneous The viscous matter present after cell lysis Step 3 should not remain Do not vortex A white flocculent precipitate of proteins cellular debris genomic DNA and detergent is formed Note When many samples are prepared in parallel mix each sample immediately after adding Precipitation Buffer E3 5 Clarify the cell lysate using centrifugation or vacuum e For Cat no 242006 clarify the lysate by centrifuging at gt 12 000 x g for 20 minutes at room temperature and transfer the particle free supernatant into a clean container Proceed directly to Removing Endotoxin page 27 e For Cat no 243006 clarify the lysate with a JetFilter as described in Steps a g below a Pour the neutralized bacterial lysate directly into the JetFilter with the yellow ring see Preparing the JetFilter page 25 b Incubate at room temperature for at least 10 minutes without agitation Note Let the lysate stand for at least 10 minutes after the transfer into the JetFilter to allow
38. ure Precipitator Module page 34 Preparing Cell Resuspending Buffer E1 Buffers and Add RNase A to the Cell Resuspending Buffer E1 Solutions according to the note with the RNase A tube Mix well The final RNase A concentration is 100 pg mL Mark the bottle label to indicate that RNase A is added Store Cell Resuspending Buffer E1 with RNase A at 4 C Lysis Buffer E2 Check the Lysis Buffer E2 for precipitates If present warm the solution briefly at 37 C to dissolve the precipitate 70 Ethanol Prepare 70 ethanol with the endotoxin free water supplied with the kit Add the appropriate amount of absolute 96 100 ethanol as stated on the label of the bottle and mix thoroughly Continued on next page 23 Gigaprep Procedure Continued Overnight Inoculate an E coli culture according to good Culture microbiological practice in growth medium with the appropriate antibiotic and grow the cells for 16 18 hours usually overnight or 24 36 hours if using a buffered growth medium e For high copy number plasmids use 2 500 mL of an overnight LB culture per sample e For low copy number plasmids use 5 000 mL of an overnight LB culture per sample Preparing the 1 Attach a JetStar DNA Binding Cartridge with a red DNA binding ring onto a clean 1 000 mL Stericup receiver flask or a Cartridge 1 Liter bottle with 45 mm neck Do not overtighten the cartridge on the bottle neck since the cartridge plasti
39. y e gt Vaccination e Automated and manual DNA sequencing e Cloning e in vitro transcription e Restriction digestion Methods Before Starting Introduction Plasmid Yields Plasmid Type and Copy Number Review the information in this section before starting Guidelines are included for growing the overnight cell culture and for determining the appropriate amounts of starting material based on the plasmid copy number used TM Some buffers in the JetStar Endotoxin free Plasmid Purification Kits contain hazardous chemicals Always wear a laboratory coat disposable gloves and eye protection when handling the buffers Important To avoid the possibility of implosion do not use vessels that are not designed for use with vacuum Do not use vessels that are cracked or scratched Always wear safety glasses when working near a bottle or flask under vacuum All types and sizes of plasmid DNA can be prepared but yields depend on the type of plasmid copy number the bacterial strain and the volume of bacterial culture used Use a high copy number plasmid to obtain a good yield of purified plasmid DNA If you use a low copy number plasmid use a higher volume of cell culture as directed in the protocol High copy number plasmids typically yield gt 3 5 ug DNA mL LB culture grown overnight Low copy number plasmids typically yield 0 2 1 ug DNA mL LB culture grown overnight Although the JetStar
Download Pdf Manuals
Related Search