EZNA® Endo-Free Plasmid DNA Midi Kit EZNA - Omega Bio-Tek
Contents
1. 21 22 23 24 25 26 27 28 29 18 Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the collection tube Add 3 mL EHB Buffer Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the collection tube Add 3 5 mL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see the instructions in the Preparing Reagents section on Page 6 Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the collection tube Repeat Steps 24 26 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind DNA Midi Column at 4 000 x g for 10 minutes to dry the column matrix Note It is important to dry the HiBind DNA Midi Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Midi Column to a nuclease free 15 mL centrifuge tube Endo Free Plasmid DNA Midi Kit Centrifugation Protocol 30 31 32 33 Add 0 5 1 mL Endo Free Elution Buffer directly to the center of the column matrix Let it sit at room temperature for 3 minutes Centrifuge at 4 000 x g for 5 minutes Note This represents approximately 65 80 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration Alternatively a second elution may be performed using the first eluate to maintain a high DNA concentration Stor2. 4 000 x g for 10 minutes at room temperature Decant or aspirate and discard the culture media Note To ensure that all traces of the medium are removed use a clean paper towel to blot excess liquid from the wall of the bottle Add 10 mL Solution I RNase A Vortex or pipet up and down to mix thoroughly Complete resuspension of cell pellet is vital for obtaining good yields Note RNase A must be added to Solution before use Please see the instructions in the Preparing Reagents section on Page 6 Add 10 mL Solution II Invert and rotate the tube gently 8 10 times to obtain a cleared lysate This may require a 2 3 minute incubation at room temperature with occasional mixing Note Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity Do not allow the lysis reaction to proceed more than 5 minutes Store Solution II tightly capped when not in use to avoid acidification from CO in the air Add 5 mL cold N3 Buffer Gently invert 10 times or until a flocculent white precipitate forms This may require a 2 minute incubation at room temperature with occasional mixing Note The solution must be mixed thoroughly This is vital for obtaining good yields If the mixture still appears viscous brownish or conglobated more mixing is required to completely neutralize the solution Prepare a Lysate Clearance Filter Syringe by removing the plunger Place the barrel in a tube rack to keep upright Make sure the e
3. Column 27 Endo Free Plasmid DNA Maxi Kit Centrifugation Protocol 18 19 20 21 22 23 24 25 26 27 28 29 30 28 Add 10 mL ETR Wash Buffer Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the collection tube Add 10 mL EHB Buffer Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the collection tube Add 15 mL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see the instructions in the Preparing Reagents section on Page 6 Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the collection tube Add 10 mL DNA Wash Buffer Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the collection tube Centrifuge the empty HiBind DNA Maxi Column at 4 000 x g for 10 minutes to dry the column matrix Note It is important to dry the HiBind DNA Maxi Column matrix before elution Residual ethanol may interfere with downstream applications Endo Free Plasmid DNA Maxi Kit Centrifugation Protocol 31 32 33 34 35 Transfer the HiBind DNA Maxi Column to a nuclease free 50 mL centrifuge tube Add 1 5 3 mL Endo Free Elution Buffer directly to the center of the column matrix Let it sit at room temperature for 5 minutes Centrifuge at 4 000 x g for 5 minutes Note This represents approximately 65 80 of bound DNA An optional s
4. Column to a 15 mL Collection Tube provided Centrifuge the empty HiBind DNA Midi Column at 4 000 x g for 10 minutes to dry the column matrix Note It is important to dry the HiBind DNA Midi Column matrix before elution Residual ethanol may interfere with downstream applications 13 31 32 33 34 35 14 Endo Free Plasmid DNA Midi Kit Vacuum Protocol Transfer the HiBind DNA Midi Column to a nuclease free 15 mL centrifuge tube Add 0 5 1 mL Endo Free Elution Buffer directly to the center of the column matrix Let it sit at room temperature for 3 minutes Centrifuge at 4 000 x g for 5 minutes Note This represents approximately 65 80 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration Alternatively a second elution may be performed using the first eluate to maintain a high DNA concentration Store DNA at 20 C Endo Free Plasmid DNA Midi Kit Centrifugation Protocol E Z N A Endo Free Plasmid DNA Midi Kit Centrifugation Protocol The following protocol is designed for plasmid DNA isolation when a vacuum manifold is not available All centrifugation steps should be performed with a swing bucket rotor for maximum plasmid DNA yields All centrifugation steps should be carried out at room temperature If high yields of low copy number plasmid DNA are desired see Low Copy Number Plasmids and Cosmids protocol on Page 31 Materials and Equipment
5. Sigma AldrichVM20 Promega Vacman or manifold with standard Luer connector B Vacuum Flask C Vacuum Tubing D Vacuum Source review tables below for pressure settings Manifold Recommended Pressure mbar VAC 08 200 to 600 Torts Tort Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Vacuum Setup age se kaso Omega Bio tek s VAC 08 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask Recommended Settings Growth and Culture of Bacteria Bacterial Strain Selection It is strongly recommended that an end A negative strain of E coli be used for routine plasmid isolation Examples of such strains include DH5a DH1 and C600 These host strains yield high quality DNA with E Z N A Endo Free Plasmid DNA Kit Protocols XL1 Blue although a slower growing strain is also recommended due to its yield of high quality DNA Host strains derivatives from HB101 such as TG1 and the JM100 series release large amounts of carbohydrates during lysis which may inhibit enzyme activity when not completely removed Some strains may also lower DNA quality due to having high levels of endonuclease activity and therefore are not recommended i e JM101 JM110 HB101 One may reduce the amount of culture volume or double the volumes of Solution Solution Il and N3 Buffer if problems are encountered with strains such as TG1 and Top10F Inoculation Bacterial cultures for pl
6. 2 Add an equal volume ETR Binding Buffer Vortex to mix thoroughly 3 Transfer the sample to the HiBind DNA Column Draw the sample through the membrane by centrifugation or vacuum 4 Continue the appropriate protocol beginning with the DNA Wash Buffer step and finishing with the elution step 32 Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the technical support staff toll free at 1 800 832 8896 Possible Problems and Suggestions Low DNA Yields Reduce the initial volume of culture or increase the lysis time while monitoring the lysis visually Cells may not have been dispersed adequately prior to the addition of Solution II Make sure to vortex cell suspension to completely disperse Solution Il if not tightly closed may need to be replaced Poor cell lysis Do not incubate cultures for more than 16 hours at 37 C Storage of cultures for extended periods prior to plasmid isolation is detrimental If using endotoxin free water for elution the pH must be Low elution efficiency 8 0 Such plasmids may yield as little as 0 1 ug plasmid DNA Low copy number from a 1 mL overnight culture plasmid used Double culture volume and follow the Low Copy Number Plasmid and Cosmid DNA Protocol Bacterial culture is overgrown or not fresh Columns were spun in a fixed angle rotor or with insufficient g force For the Midi and Maxi kit
7. 6 Solution 250 mL Solution Il 250 mL N3 Buffer 135 mL ETR Binding Buffer 650 mL ETR Wash Buffer 270 mL DNA Wash Buffer 100 mL RNase A 5 mL Endo Free Elution Buffer 100 mL HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and Vacman are all trademarks of their respective companies PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 35
8. Ca OMEGA Innovations in nucleic acid isolation Product Manual E Z N A Endo Free Plasmid DNA Midi Kit D6915 00 2 preps D6915 01 10 preps D6915 03 25 preps D6915 04 100 preps E Z N A Endo Free Plasmid DNA Maxi Kit D6926 00 2 preps D6926 01 6 preps D6926 03 25 preps D6926 04 100 preps April 2013 For research use only Not intended for diagnostic testing E Z N A Endo Free Plasmid DNA Midi Kit E Z N A Endo Free Plasmid DNA Maxi Kit Table of Contents IntrodUcti ON nana EAN 2 Yield and Quality GONE essences 3 Illustrated Protocol Snine mannan 4 Kit CONTENTS sssssisr ecserin n rees Er R EEEIEE R ER OEI SETTIR ER AEE ESETERE 5 Preparing Reagents Storage and Stability 6 Guidelines for Vacuum MAnifoldhssssssessensssenntenunsse 7 Endo Free Plasmid DNA Midi Vacuum Protocol 10 Endo Free Plasmid DNA Midi Centrifugation Protocol 15 Endo Free Plasmid DNA Maxi Vacuum Protocol 20 Endo Free Plasmid DNA Maxi Centrifugation Protocol 25 Mode eo etecenerecesdeureresecesteteecteeeenactescneee 30 Troubleshooting Guide minima 33 Orne era 35 Manual Revision April 2013 04 OMEGA bio tek Innovations in nucleic acid isolation Introduction The E Z N A Endo Free Plasmid Midi Kit and the Endo Free Plasmid Maxi Kit combine the power of HiBind technology with Omega Bio tek s innovative Endotoxin Removal Technology ETR to deliver high quality plasmid DNA with low endotoxin level
9. Page 31 Materials and Equipment to be Supplied by User 100 Ethanol Do not use denatured alcohol e Centrifuge with swing bucket rotor capable of at least 4 000 x g See the Troubleshooting section on Page 27 if not available e Vortexer e Nuclease free 50 mL centrifuge tubes Appropriate centrifuge bottle for Step 1 Ice bucket e Optional Water bath or incubator capable of 70 C e Optional 3M NaOH Before starting Chill N3 Buffer on ice Heat Elution Buffer to 70 C if Plasmid DNA is gt 10 kb Prepare DNA Wash Buffer and Solution 1 according to Preparing Reagents section on Page 6 1 Transfer 50 200 mL overnight culture to an appropriate centrifuge bottle Note The optimal volume to use depends on the culture density and plasmid copy number The optimal cell mass OD x mL culture for the HiBind DNA Maxi Column is 300 400 For example if the OD of a culture is 4 0 the optimal culture volume should be 75 100 mL If excess culture cell mass is used alkaline lysis will be ineffi cient the HiBind membrane will be overloaded and the performance of the system will be decreased The increase in lysate viscosity will require vigorous mixing which may result in shearing of genomic DNA and contamination the plasmid DNA For low copy number plasmids see the Low Copy Number Plasmids protocol on Page 31 25 Endo Free Plasmid DNA Maxi Kit Centrifugation Protocol 26 Centrifuge at
10. Transfer the HiBind DNA Maxi Column to a 50 mL Collection Tube provided 23 32 33 34 35 36 37 24 Endo Free Plasmid DNA Maxi Kit Vacuum Protocol Centrifuge the empty HiBind DNA Maxi Column at 4 000 x g for 10 minutes to dry the column matrix Note It is important to dry the HiBind DNA Maxi Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Maxi Column to a nuclease free 50 mL centrifuge tube Add 1 5 3 mL Endo Free Elution Buffer directly to the center of the column matrix Let it sit at room temperature for 5 minutes Centrifuge at 4 000 x g for 5 minutes Note This represents approximately 65 80 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration Alternatively a second elution may be performed using the first eluate to maintain a high DNA concentration Store DNA at 20 C Endo Free Plasmid DNA Maxi Kit Centrifugation Protocol E Z N A Endo Free Plasmid DNA Maxi Kit Centrifugation Protocol The following protocol is designed for plasmid DNA isolation when a vacuum manifold is not available All centrifugation steps should be performed with a swing bucket rotor for maximum plasmid DNA yields All centrifugation steps should be carried out at room temperature If high yields of low copy number plasmid DNA are desired see Low Copy Number Plasmids and Cosmids protocol on
11. arance Filter Syringe Hold the Lysate Clearance Filter Syringe barrel over a clean 15 mL centrifuge tube not provided and remove the end cap from the syringe tip Endo Free Plasmid DNA Midi Kit Centrifugation Protocol 10 Gently insert the plunger into the barrel to expel the cleared lysate into the 15 mL centrifuge tube Note Some of the lysate may remain in the flocculent precipitate DO NOT force this residual lysate through the filter 11 Measure the volume of cleared lysate 12 Add 1 volume ETR Binding Buffer Invert the tube gently 10 times Note Steps 13 32 should be performed in a swing bucket rotor for maximum plasmid DNA yield All of centrifugation steps should be carried out at room temperature 13 Insert a HiBind DNA Midi Column into a 15 mL Collection Tube provided Optional Protocol for Column Equilibration Add 1 mL 3M NaOH to the HiBind DNA Midi Column Let sit at room temperature for 4 minutes Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the collection tube FUN 14 Transfer 3 5 mL cleared supernatant from Step 12 to the HiBind DNA Midi Column 15 Centrifuge at 4 000 x g for 3 minutes 16 Discard the filtrate and reuse the collection tube 17 Repeat Steps 14 16 until all of the cleared supernatant has been transferred to the HiBind DNA Midi Column 18 Add 2 mL ETR Wash Buffer 17 Endo Free Plasmid DNA Midi Kit Centrifugation Protocol 19 20
12. asmid preparations should always be grown from a single colony picked from a freshly streaked plate Subculturing directly from glycerol stock or liquid cultures may lead to uneven yields or plasmid loss Optimal results are obtained by using one single isolated colony from a freshly transformed or freshly streaked plate to inoculate an appropriate volume of starter culture containing the appropriate antibiotic and then incubated for 12 16 at 37 C with vigorous shaking 300 rpm shaking incubator Note Aeration is very important The culture volume should not exceed 1 4 the volume of the container Culture Media The E Z N A Endo Free Plasmid DNA Kits are specially designed for use with cultures grown in Luria Bertani LB medium Richer broths such as TB Terrific Broth or 2 x YT lead to high cell densities that can overload the purification system and therefore are not recommended If rich media has to be used growth times have to be optimized and the recommended culture volumes must be reduced to match the capacity of the HiBind DNA Column Note As culture ages DNA yield may begin to decrease due to cell death and lysis within the culture Recommended Settings Culture Volume and Cell Density Do Not Exceed Maximum Recommended Culture Volumes For optimal plasmid yields the starting culture volume should be based on culture cell density A bacterial density between 2 0 and 3 0 at OD is recommended When using nutrient rich m
13. axi Columns 2 e 25 10 50 mL Collection Tubes 2 6 2 100 Lysate Clearance Filter Syringe 2 6 2 Solution 1050 mL Solution II 1050 mL ETR Binding Buffer 3 x 900 mL ETR Wash Buffer 2x 550 mL EHB Buffer 1050 mL DNA Wash Buffer 3 x 200 mL Endo Free Elution Buffer 2x 160 mL Preparing Reagents 1 Add vial of RNase A to the bottle of Solution provided and store at 2 8 C 2 Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature ek 10006 Ethanol tobe Added kit 00 Ethanoltobe nade 3 Check Solution II EHB Buffer and ETR Binding Buffer for precipitates before use Redissolve any precipitates by warming to 37 C Storage and Stability All of the E Z N A Endo Free Plasmid DNA Midi and Maxi Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows After RNase A is added Solution should be stored at 2 8 C All other components should be stored at room temperature Store Solution II tightly capped when not in use During shipment or storage in cool ambient conditions precipitates may form in EHB Buffer Solution II and ETR Binding Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking Guidelines for Vacuum Manifold The following is required for use with the Vacuum Spin Protocol A Vacuum Manifold We recommend Omega Bio tek s VAC 08 Other Compatible Vacuum Manifolds Qiagen QlAvac24
14. bacteria use the following modified protocol Note The E Z N A Endo Free Plasmid DNA Midi Kit and the E Z N A Endo Free Plasmid DNA Maxi Kit come with enough buffers to perform the standard protocols Additional buffers are needed to perform the Low Copy Number Plasmid and Cosmid DNA Protocol These buffers can be purchased separately See Page 35 for ordering information 1 Increase the volume of starting culture from that of high copy number plasmids Use 50 100 mL bacterial culture for the E Z N A Endo Free Plasmid DNA Midi Kit and 200 400 mL bacterial culture for the E Z N A Endo Free Plasmid DNA Maxi Kit 2 Pellet the bacterial cells by centrifugation 3 Decant or aspirate and discard the culture media 4 Perform Steps 4 12 in the standard protocols with double volumes of Solution Solution II N3 Buffer and ETR Binding Buffer 5 Continue with Step 13 of the standard protocols by following the wash drying and elution steps There is no need to increase the volumes of EHB Buffer ETR Wash Buffer DNA Wash Buffer or Endo Free Elution Buffer 31 Purification of Plasmid DNA prepared by other Methods E Z N A Endo Free Plasmid DNA Midi Maxi Kit Protocol Plasmid DNA Purification Plasmid DNA isolated using other methods can be further purified with the following protocol The protocol includes the removal of endotoxins from the sample 1 Bring the volume of plasmid DNA to 2 mL with sterile deionized water
15. dd 1 25 mL cold N3 Buffer Gently invert 10 times or until a flocculent white precipitate forms This may require a 2 minute incubation at room temperature with occasional mixing Note The solution must be mixed thoroughly This is vital for obtaining good yields If the mixture still appears viscous brownish or conglobated more mixing is required to completely neutralize the solution Prepare a Lysate Clearance Filter Syringe by removing the plunger Place the barrel in a tube rack to keep upright Make sure the end cap is attached to the syringe tip Immediately transfer the lysate from Step 6 into the barrel of the Lysate Clearance Filter Syringe Hold the Lysate Clearance Filter Syringe barrel over a clean 15 mL centrifuge tube not provided and remove the end cap from the syringe tip 11 10 11 12 13 14 Endo Free Plasmid DNA Midi Kit Vacuum Protocol Gently insert the plunger into the barrel to expel the cleared lysate into the 15 mL centrifuge tube Note Some of the lysate may remain in the flocculent precipitate DO NOT force this residual lysate through the filter Measure the volume of cleared lysate Add 1 volume ETR Binding Buffer Invert the tube gently 10 times Prepare the vacuum manifold by following the manufacturer s instructions Connect the HiBind DNA Midi Column to the vacuum manifold Refer to the Illustrated Vacuum Set Up on Page 7 for details Optional Protocol for Column Equi
16. e DNA at 20 C 19 Endo Free Plasmid DNA Maxi Kit Vacuum Protocol E Z N A Endo Free Plasmid DNA Maxi Kit Vacuum Protocol All centrifugation steps should be performed with a swing bucket rotor for maximum plasmid DNA yields All centrifugation steps should be carried out at room temperature If high yields of low copy number plasmid DNA are desired see Low Copy Number Plasmids and Cosmids protocol on Page 31 Materials and Equipment to be Supplied by User 100 Ethanol Do not use denatured alcohol e Centrifuge with swing bucket rotor capable of at least 4 000 x g See the Troubleshooting section on Page 27 if not available Vacuum manifold Vortexer Nuclease free 50 mL centrifuge tubes Appropriate centrifuge bottle for Step 1 e Ice bucket e Optional Water bath or incubator capable of 70 C e Optional 3M NaOH Before starting Chill N3 Buffer on ice Heat Elution Buffer to 70 C if Plasmid DNA is gt 10 kb Prepare DNA Wash Buffer and Solution 1 according to Preparing Reagents section on Page 6 1 Transfer 50 200 mL overnight culture to an appropriate centrifuge bottle not provided Note The optimal volume to use depends on the culture density and plasmid copy number The optimal cell mass OD x mL culture for the HiBind DNA Maxi Column is 300 400 For example if the OD of a culture is 4 0 the optimal culture volume should be 75 100 mL If excess culture ce
17. econd elution will yield any residual DNA though at a lower concentration Alternatively a second elution may be performed using the first eluate to maintain a high DNA concentration Store DNA at 20 C 29 DNA Precipitation The concentration of the eluted plasmid DNA varies with copy number host strain and growth conditions In some cases residual ethanol may also be present To adjust the DNA concentration following plasmid DNA elution or for the removal of residual ethanol perform the following isopropanol precipitation protocol 1 Carefully transfer the eluted plasmid DNA to a clean tube suitable for precipitation Add 1 10 volume 3M NaAC pH 5 2 and 0 7 volume isopropanol room temperature Vortex to mix 2 Centrifuge at gt 15 000 x g for 20 minutes at 4 C 3 Carefully decant the supernatant 4 Add 1 2 mL 70 ethanol Vortex to resuspend the pellet 5 Centrifuge at gt 15 000 x g for 10 minutes at 4 C 6 Carefully decant the supernatant 7 Air dry the pellet for 10 minutes 8 Add 200 500 uL Elution Buffer 9 Store DNA at 20 C 30 Low Copy Number Plasmid and Cosmid DNA Purification E Z N A Endo Free Plasmid DNA Midi Maxi Kit Protocol Low Copy Number Plasmid and Cosmid DNA Protocol Low copy number plasmids generally give 0 1 1 ug DNA per mL overnight culture For the isolation of plasmid DNA from low copy number plasmids 0 1 1 ug mL culture or low copy number plasmid 1 2 g mL culture
18. edia care should be taken to ensure that the cell density does not exceed an OD of 3 0 Using a high density culture outside of the recommended OD range may overload the purification system Endo Free Plasmid DNA Midi Kit Vacuum Protocol E Z N A Endo Free Plasmid DNA Midi Kit Vacuum Protocol All centrifugation steps should be performed with a swing bucket rotor for maximum plasmid DNA yields All centrifugation steps should be carried out at room temperature If high yields of low copy number plasmid DNA are desired see Low Copy Number Plasmids and Cosmids protocol on Page 31 Materials and Equipment to be Supplied by User 100 Ethanol Do not use denatured alcohol e Centrifuge with swing bucket rotor capable of at least 4 000 x g See the Troubleshooting section on Page 34 if not available Nuclease free 15 mL and 50 mL centrifuge tubes Vacuum manifold e Vortexer e Ice bucket e Optional Water bath or incubator capable of 70 C e Optional 3M NaOH Before starting e Chill N3 Buffer on ice Heat Elution Buffer to 70 C if Plasmid DNA is gt 10 kb Prepare DNA Wash Buffer and Solution 1 according to Preparing Reagents section on Page 6 1 Transfer 20 50 mL overnight culture to a 50 mL centrifuge tube not provided Note The optimal volume to use depends on the culture density and plasmid copy number The optimal cell mass OD x mL culture for the HiBind DNA Midi Colum
19. following table Sample yields from 50 mL starting culture Expected Yield rame in copy Number 50 mL culture pBluescript vectors ColE14 300 500 100 180 ug pGEM vectors 300 400 100 200 pg Centrifugation Protocol Q 1 Pellet by Centrifugation Resuspend Lyse Neutralize Transfer Lysate to Syringe Insert Plunger into Syringe and Clear Lysate into 15 mL Collection Tube Add ETR Binding Buffer Add Equilibration Buffer Pass Lysate through HiBind DNA Column Wash 4X Dry HiBind matrix Elute Endotoxin free DNA Vacuum Protocol QC Pellet by Centrifugation Resuspend Lyse Neutralize Transfer Lysate to Syringe Insert Plunger into Syringe and Clear Lysate into 15 mL Collection Tube Prepare Vacuum Manifold Add ETR Binding Buffer Insert column into leur connector on vacuum manifold Add Equilibration Buffer Pass Lysate through HiBind DNA Column Wash 4X Dry HiBind matrix Elute Endotoxin free DNA Kit Contents E Z N A Endo Free Plasmid DNA Midi Kit D6915 04 HiBind DNA Midi Columns 100 15 mL Collection Tubes 100 Lysate Clearance Filter Syringe 100 Solution 270 mL Solution Il 270 mL ETR Binding Buffer 650 mL ETR Wash Buffer 230 mL EHB Buffer 330 mL DNA Wash Buffer 200 mL Endo Free Elution Buffer 200 mL E Z N A Endo Free Plasmid DNA Maxi Kit D6926 04 Purifications mee e 100 HiBind DNA M
20. h this kit An optional Column Equilibration Protocol has been added to the protocol for your convenience e Equilibration Buffer is replaced with 3M NaOH provided by the user Yield and Quality of DNA Determine the absorbance of an appropriate dilution 20 to 50 fold of the sample at 260 nm and then at 280 nm The DNA concentration is calculated as follows DNA concentration Absorbance 260 x 50 x Dilution Factor pg mL A ratio greater than 1 8 indicates greater than 90 nucleic acid Alternatively quantity as well as quality can sometimes best be determined by agarose gel ethidium bromide electrophoresis by comparison to DNA samples of known concentrations Typically the majority of the DNA eluted is in monomeric supercoil form though concatamers may also be present Plasmid Copy Number and Expected Yield The yield and quality of the plasmid DNA obtained depends on a number of factors including plasmid copy number size of insert host strain culture volume culture medium and binding capacity of the kits Of these factors the vector copy number culture volume and kit binding capacity are most important Plasmid copy number ranges from one copy to several hundred copies per cell as dictated by their origin of replication But very large plasmids often display a very low copy number per cell The expected yield of 50 mL overnight cultures LB medium with the E Z N A Endo Free Plasmid DNA Midi or Maxi Kit are indicated in the
21. i Column Let sit at room temperature for 5 minutes Turn on the vacuum source to draw the buffer through the column Turn off the vacuum PWNS Transfer the cleared lysate from Step 12 to the HiBind DNA Maxi Column Turn on the vacuum source to draw the lysate through the column Continue adding lysate into until you have 500 uL lysate remaining in the column Note Leaving the 500 uL lysate in the column prevents foaming in the following steps and allows for faster processing See the illustration below L Leaving 500 uL solution in the column after Step 21 reduces foam which decreases total processing RE O time and improves efficiency 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Endo Free Plasmid DNA Maxi Kit Vacuum Protocol Turn off the vacuum Add 10 mL ETR Wash Buffer Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Add 10 mL EHB Buffer Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Add 15 mL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see the instructions in the Preparing Reagents section on Page 6 Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Add 10 mL DNA Wash Buffer Turn on the vacuum source to draw the buffer through the column Turn off the vacuum
22. libration 15 16 17 12 1 Add 1 mL3M NaOH to the HiBind DNA Midi Column 2 Turn on the vacuum source to draw the buffer through the column 3 Turnoffthe vacuum Transfer the cleared lysate from Step 13 to the HiBind DNA Midi Column Turn on the vacuum source to draw the lysate through the column Continue adding lysate into until you have 500 uL lysate remaining in the column Note Leaving the 500 uL lysate in the column prevents foaming in the following steps and allows for faster processing See the illustration below E Leaving 500 uL solution in the column after Step 21 reduces foam which decreases total processing T time and improves efficiency p It 7 m 18 19 20 21 22 23 24 25 26 27 28 29 30 Endo Free Plasmid DNA Midi Kit Vacuum Protocol Turn off the vacuum Add 2 mL ETR Wash Buffer Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Add 3 mL EHB Buffer Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Add 3 5 mL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see the instructions in the Preparing Reagents section on Page 6 Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Repeat Steps 25 27 for a second DNA Wash Buffer wash step Transfer the HiBind DNA Midi
23. ll mass is used alkaline lysis will be ineff cient the HiBind membrane will be overloaded and the performance of the system will be decreased The increase in lysate viscosity will require vigorous mixing which may result in shearing of genomic DNA and contamination the plasmid DNA For low copy number plasmids see the Low Copy Number Plasmids protocol on Page 31 20 Endo Free Plasmid DNA Maxi Kit Vacuum Protocol Centrifuge at 4 000 x g for 10 minutes at room temperature Decant or aspirate and discard the culture media Note To ensure that all traces of the medium are removed use a clean paper towel to blot excess liquid from the wall of the bottle Add 10 mL Solution I RNase A Vortex or pipet up and down to mix thoroughly Complete resuspension of cell pellet is vital for obtaining good yields Note RNase A must be added to Solution before use Please see the instructions in the Preparing Reagents section on Page 6 Add 10 mL Solution II Invert and rotate the tube gently 8 10 times to obtain a cleared lysate This may require a 2 3 minute incubation at room temperature with occasional mixing Note Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity Do not allow the lysis reaction to proceed more than 5 minutes Store Solution ll tightly capped when not in use to avoid acidification from CO in the air Add 5 mL cold N3 Buffer Gently invert 10 times or until a flocculent white p
24. n is 80 100 For example if the OD of a culture is 4 0 the optimal culture volume should be 20 25 mL If excess culture cell mass is used alkaline lysis will be inefficient the HiBind membrane will be overloaded and the performance of the system will be decreased The increase in lysate viscosity will require vigorous mixing which may result in shearing of genomic DNA and contamination the plasmid DNA For low copy number plasmids see the Low Copy Number Plasmids protocol on Page 31 2 Centrifuge at 4 000 x g for 10 minutes at room temperature 10 Endo Free Plasmid DNA Midi Kit Vacuum Protocol Decant or aspirate and discard the culture media Note To ensure that all traces of the medium are removed use a clean paper towel to blot excess liquid from the wall of the tube Add 2 5 mL Solution I RNase A Vortex or pipet up and down to completely resuspend the cells Note RNase A must be added to Solution before use Please see the instructions in the Preparing Reagents section on Page 6 Add 2 5 mL Solution II Invert and rotate the tube gently 8 10 times to obtain a cleared lysate This may require a 2 3 minute incubation at room temperature with occasional mixing Note Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity Do not allow the lysis reaction to proceed more than 5 minutes Store Solution Il tightly capped when not in use to avoid acidification from CO in the air A
25. nd cap is attached to the syringe tip Immediately transfer the lysate from Step 6 into the barrel of the Lysate Clearance Filter Syringe Hold the Lysate Clearance Filter Syringe barrel over a new 50 mL centrifuge tube not Endo Free Plasmid DNA Maxi Kit Centrifugation Protocol provided and remove the end cap from the syringe tip 10 Gently insert the plunger into the barrel to expel the cleared lysate into the 50 mL centrifuge tube Note Some of the lysate may remain in the flocculent precipitate DO NOT force this residual lysate through the filter 11 Measure the volume of cleared lysate 12 Add 1 volume ETR Binding Buffer Invert the tube gently 10 times Note Steps 13 35 should be performed in a swing bucket rotor for maximum plasmid DNA yield All of centrifugation steps should be carried out at room temperature 13 Insert a HiBind DNA Maxi Column into a 50 mL Collection Tube provided Optional Protocol for Column Equilibration Add 3 mL 3M NaOH to the HiBind DNA Maxi Column Let sit at room temperature for 4 minutes Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the collection tube FWN gt 14 Transfer 20 mL cleared supernatant from Step 12 to the HiBind DNA Maxi Column 15 Centrifuge at 4 000 x g for 3 minutes 16 Discard the filtrate and reuse the collection tube 17 Repeat Steps 14 16 until all of the cleared supernatant has been transferred to the HiBind DNA Maxi
26. nd discard the culture media Note To ensure that all traces of the medium are removed use a clean paper towel to blot excess liquid from the wall of the tube Add 2 5 mL Solution I RNase A Vortex or pipet up and down to completely resuspend the cells Note RNase A must be added to Solution before use Please see the instructions in the Preparing Reagents section on Page 6 Add 2 5 mL Solution II Invert and rotate the tube gently 8 10 times to obtain a cleared lysate This may require a 2 3 minute incubation at room temperature with occasional mixing Note Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity Do not allow the lysis reaction to proceed more than 5 minutes Store Solution II tightly capped when not in use to avoid acidification from CO in the air Add 1 25 mL cold N3 Buffer Gently invert 10 times or until a flocculent white precipitate forms This may require a 2 minute incubation at room temperature with occasional mixing Note The solution must be mixed thoroughly This is vital for obtaining good yields If the mixture still appears viscous brownish or conglobated more mixing is required to completely neutralize the solution Prepare a Lysate Clearance Filter Syringe by removing the plunger Place the barrel in a tube rack to keep upright Make sure the end cap is attached to the syringe tip Immediately transfer the lysate from Step 6 into the barrel of the Lysate Cle
27. recipitate forms This may require a 2 minute incubation at room temperature with occasional mixing Note The solution must be mixed thoroughly This is vital for obtaining good yields If the mixture still appears viscous brownish or conglobated more mixing is required to completely neutralize the solution Prepare a Lysate Clearance Filter Syringe by removing the plunger Place the barrel in a tube rack to keep upright Make sure the end cap is attached to the syringe tip Immediately transfer the lysate from Step 6 into the barrel of the Lysate Clearance Filter Syringe Hold the Lysate Clearance Filter Syringe barrel over a new 50 mL centrifuge tube not 21 10 11 12 13 14 Endo Free Plasmid DNA Maxi Kit Vacuum Protocol provided and remove the end cap from the syringe tip Gently insert the plunger into the barrel to expel the cleared lysate into the 50 mL centrifuge tube Note Some of the lysate may remain in the flocculent precipitate DO NOT force this residual lysate through the filter Measure the volume of cleared lysate Add 1 volume ETR Binding Buffer Invert the tube gently 10 times Prepare the vacuum manifold by following the manufacturer s instructions Connect the HiBind DNA Maxi Column to the vacuum manifold Refer to the Illustrated Vacuum Set Up on Page 7 for details Optional Protocol for Column Equilibration 15 16 17 22 Add 3 mL 3M NaOH to the HiBind DNA Mid
28. s the columns must be spun in a swing bucket rotor at 4 000 x g for liquids to pass through efficiently Alkaline lysis is Reduce the lysis time Solution II to 3 minutes or until prolonged the suspended cells form a clear viscous solution Confirm the cell density by measuring OD To calculate Too many or too few the volume of culture to use take the desired cell mass cells were used and divide by the absorbance of the overnight culture at 600 nm No DNA Eluted DNA Wash Buffer not Prepare DNA Wash Buffer according to the instructions diluted with ethanol on Page 6 High molecular weight DNA contamination of product Over mixing of cell oe Do not vortex or mix aggressively after adding Solution lysate upon addition of Il Solution Il e Overgrown culture contain lysed cells and degraded Culture overgrown DNA Do not grow cell longer than 16 hours 33 Troubleshooting Guide Plasmid DNA floats out of well while loading agarose gel Ethanol has not been removed completely from column following wash steps Centrifuge column as instructed to dry the column before elution Incubate columns for 10 minutes at 65 C to completely dry membrane after centrifugation step Absorbance of purified DNA does not accurately reflect quality of the ratio is too high or too low plasmid A DNA Wash Buffer is diluted with ethanol containing impurities Plasmid DNA is contaminated with RNA RNase A trea
29. s for use in eukaryotic transfection and in vitro experiments Endotoxins are lipopolysaccharides LPS found in the outer cell membrane of gram negative bacteria such as E coli One E coli cell contains around 2 million LPS molecules each having hydrophobic hydrophilic and charged regions Bacteria release small quantities of endotoxins during growth and large quantities at death At the time of lysis during plasmid purification endotoxins are shed into the lysate The chemical and physical properties that endotoxin molecules possess lead to their co purification with plasmid DNA by behaving similarly on the surface of silica and anion exchange resins The E Z N A Endo Free Plasmid System uses a specially formulated buffer that prevents endotoxin molecules from binding to the surface of the HiBind matrix In addition the E Z N A Endo Free Plasmid Midi and Maxi Kits include specialized filter cartridges that replace the centrifugation step following alkaline lysis For the best interpretation of results it is crucial that the purified plasmid DNA be free of endotoxins Endotoxin contamination lowers transfection efficiencies for endotoxin sensitive cell lines For gene therapy endotoxin contamination should be of major concern since endotoxins have the potential to cause fever endotoxic shock syndrome and interfere with in vitro transfection into immune cells New In this Edition e Equilibration Buffer is no longer included wit
30. tment is insufficient Background reading is high due to fine silica particulates Purification is incomplete due to column overloading Plasmid DNA is contaminated with chromosomal DNA 260 Check the absorbance of the ethanol between 250 nm and 300 nm Do not use ethanol with high absorbance Trace impurities may remain on the column after washing and can contribute to the absorbance Confirm that the RNase A was added to Solution I prior to first use The RNase A Solution may degrade due to high temperatures gt 65 C or prolonged storage gt 6 months at room temperature Spin the DNA sample at maximum speed for 1 minute use the supernatant to repeat the absorbance readings Reduce the initial volume of culture Do not use cultures that have grown for more than 24 hours or are in the cell death phase Do not vortex or vigorously shake the cells during the lysis reaction or after adding N3 Buffer 4 000 x g centrifuge not available 4 000 x g centrifuge not available 34 For centrifuges only capable of 2 000 4 000 x g increase all centrifugation times by 2 minutes except for the drying of the column Increase drying by 5 minutes It may be necessary to incubate the empty column for drying step at 65 C for 10 minutes to completely dry the column A Swing Bucket Centrifuge is Required Ordering Information The following components are available for purchase separately Call Toll Free at 800 832 889
31. to be Supplied by User 100 Ethanol Do not use denatured alcohol e Centrifuge with swing bucket rotor capable of at least 4 000 x g See the Troubleshooting section on Page 34 if not available e Nuclease free 15 mL and 50 mL centrifuge tubes e Vortexer e Ice bucket e Optional Water bath or incubator capable of 70 C e Optional 3M NaOH Before starting Chill N3 Buffer on ice Heat Elution Buffer to 70 C if Plasmid DNA is gt 10 kb Prepare DNA Wash Buffer and Solution 1 according to Preparing Reagents section on Page 6 1 Transfer 20 50 mL overnight culture to a 50 mL centrifuge tube not provided Note The optimal volume to use depends on the culture density and plasmid copy number The optimal cell mass OD x mL culture for the HiBind DNA Midi Column is 80 100 For example if the OD of a culture is 4 0 the optimal culture volume should be 20 25 mL If excess culture cell mass is used alkaline lysis will be inefficient the HiBind membrane will be overloaded and the performance of the system will be decreased The increase in lysate viscosity will require vigorous mixing which may result in shearing of genomic DNA and contamination the plasmid DNA For low copy number plasmids see the Low Copy Number Plasmids protocol on Page 31 2 Centrifuge at 4 000 x g for 10 minutes at room temperature 15 Endo Free Plasmid DNA Midi Kit Centrifugation Protocol 16 Decant or aspirate a
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