EZNA®Plasmid DNA Midi Kit EZNA®Plasmid DNA - Omega Bio-Tek
Contents
1. Plasmid DNA is contami 4 000 x g centrifuge not available A Swing Bucket Centrifuge is Required 29 30 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 sr Solution 250 mL Solution II 250 mL Solution Ill 250 mL Elution Buffer 100 mL PDRO48 DNA Wash Buffer 100 mL PSO10 RNase A 400 uL AC117 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 license2. 19 16 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 Wwa Transfer 3 5 mL cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind DNA Midi Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Midi Column Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the collection tube Repeat Steps 10 12 until all of the cleared supernatant has been transferred to the HiBind DNA Midi Column Add 3 mL HBC Buffer Note HBC Buffer must be diluted with isopropanol 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 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 E Z N A Plasmid DNA Midi Kit Centrifugation Protocol 20 21 22 23 24 25 26 Repeat Steps 17 19 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 d
3. 19 E Z N A Plasmid DNA Maxi Kit Vacuum Protocol Optional Protocol for Column Equilibration 11 12 13 14 T5 16 ve 18 19 20 20 Add 3 mL 3M NaOH to the HiBind DNA Maxi Column Let sit at room temperature for 4 minutes Turn on the vacuum source to draw the NaOH through the column Turn off the vacuum Saat ic Transfer 20 mL cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind DNA Maxi Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Maxi Column Turn on the vacuum source to draw the supernatant through the column Turn off the vacuum Repeat Steps 11 13 until all of the cleared supernatant has been transferred to the HiBind DNA Maxi Column Add 10 mL HBC Buffer Note HBC Buffer must be diluted with isopropanol 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 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 Repeat Steps 17 19 for a second DNA Wash Buffer wash step 21 22 23 24 25 26 E Z N A Plasmid DNA Maxi Kit Vacuum Protocol Continue to apply
4. DNA yields All of centrifugation steps should be carried out at room temperature 9 Insert a HiBind DNA Maxi Column into a 50 mL Collection Tube provided 23 E Z N A Plasmid DNA Maxi Kit Centrifugation Protocol Optional Protocol for Column Equilibration 10 11 12 13 14 15 16 ve 18 19 24 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 Www oa Transfer 20 mL cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind DNA Maxi Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Maxi Column Centrifuge at 4 000 x g for 5 minutes Discard the filtrate and reuse the collection tube Repeat Steps 10 12 until all of the cleared supernatant has been transferred to the HiBind DNA Maxi Column Add 10 mL HBC Buffer Note HBC Buffer must be diluted with isopropanol prior to use Please see the instructions in the Preparing Reagents section on Page 6 Centrifuge at 4 000 x g for 5 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 5 minutes Discard the f
5. 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 Plasmid DNA Midi or Maxi Kit are indicated in the following table Sample yields from 50 mL starting culture Expected Yield Pema O Reien copy Number 50 mt culture pBluescript vectors ColE14 300 500 100 180 ug pGEM vectors 300 400 100 200 pg Centrifugation Protocol Vacuum Protocol Pellet by Centrifugation O Pellet by Centrifugation Resuspend cal Resuspend Lyse g Lyse Neutralize a Neutralize Clear Lysate O Clear Lysate Transfer cleared lysate to g Transfer cleared lysate to HiBind DNA Column HiBind DNA Column Bind Bind a Hl Wash
6. precipitates by warming to 37 C Prepare DNA Wash Buffer and Solution 1 according to Preparing Reagents section on Page 6 Optional Heat Elution Buffer to 65 C if plasmid DNA is gt 10 kb 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 27 Centrifuge at 4 000 x g for 10 minutes at room temperature E Z N A Plasmid DNA Midi Kit Centrifugation Protocol 3 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 4 Add 2 25 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 secti
7. 0 mL centrifuge tubes capable of withstanding 15 000 x g Appropriate centrifuge bottle for Step 1 Optional Water bath incubator or heat block capable of 65 C Optional Sterile deionized water Optional 3M NaOH Before starting 22 Check Solution Il and Solution Ill for precipitation before use Redissolve any precipitates by warming to 37 C Prepare DNA Wash Buffer and Solution 1 according to Preparing Reagents section on Page 6 Optional Heat Elution Buffer to 65 C if plasmid DNA is gt 10 kb 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 cell 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 27 Centrifuge at 4 000 x g for 10 minutes at room temperature E Z N A Plasmid DNA Maxi Kit Centrifugation Protocol 3 Decant or aspirate and discard the culture media Note To ensu
8. 3X Wash 3X iS Dry Dry Elute Elute Kit Contents E Z N A Plasmid DNA Midi Kit D6904 04 Purifications 100 Hibind DNA Midi Columns 100 15 mL Collection Tubes 100 Solution 270 mL Solution II 270 mL Solution III 400 mL HBC Buffer wani DNA Wash Buffer 700 mt Elution Buffer 220 mL User Manual V E Z N A Plasmid DNA Maxi Kit D6922 00 D6922 01 D6922 02 D6922 04 Purifications 100 HiBind DNA Maxi Columns 100 50 mL Collection Tubes 100 Soliton 2675 mL Solution I 2675 mL Solution Il 3850 ml HBC Buffer 3x 250 mL DNA Wash Buffer 4x 200 mL RNase A ani Elution Buffer 2x 350 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 fet 10006 Ethanol tobe Added kit 00 Ethanoltobe nae 3 Dilute HBC Buffer with isopropanol as follows and store at room temperature et wopropanoitobenaed _ e wopropanottobenaed _ 4 Check Solution II Solution Ill and HBC Buffer for precipitation before use Redissolve any precipitation by warming to 37 C Guidelines for Vacuum Manifold The following is required for use with the Vacuum Protocol A Vacuum Manifold We recommend Omega Bio tek s VAC 08 Other Compatible Vacuum Manifolds Qiagen QlAvac24 Sigma AldrichVM20 Promega Vacman or manifold with standard luer connector B Vacuum Flask C Vacuum Tubi
9. Ca OMEGA Innovations in nucleic acid isolation 7 Product Manual E Z N A Plasmid DNA Midi Kit D6904 00 2 preps D6904 01 10 preps D6904 03 25 preps D6904 04 100 preps E Z N A Plasmid DNA Maxi Kit D6922 00 2 preps D6922 01 5 preps D6922 02 20 preps D6922 04 100 preps May 2013 For research use only Not intended for diagnostic testing E Z N A Plasmid DNA Midi Kit E Z N A Plasmid DNA Maxi Kit Table of Contents Introduction Storage and Stability sssss s mseememmemm 2 Yield and Quality of DNA iii ain 3 Illustrated ProtOColS sessessessesseessesesssssssssssrssssessnssessnssessnssssssssnsensennses ss 4 Kit SISI AA KAABA AAA ET REEI SHA 5 Preparing RAGE ii 6 Guidelines for Vacuum Manifold s ssssssesseseessrsressrsresrssssssssrsersssssess 7 Recommended SettiNGS sescssscssecsescssecseesssccseeseecsecesseeseecseeeseesseesseeses 8 Plasmid DNA Midi Kit Vacuum ProtoCol sssseseesesseeseseesseeseereeee 10 Plasmid DNA Midi Kit Centrifugation Protocol 14 Plasmid DNA Maxi Kit Vacuum PrOotocoll secscssccssecssecseesees 18 Plasmid DNA Maxi Kit Centrifugation ProtocOl eev lt sssssssssevecs 22 DNA PrecipitatiOn ssessssessccsooessessssesrceesssesssrsssusneeesseeseceossusecoosseeeorress 26 Low Copy Number Plasmids and Cosmid6s ssssssscssecssecserseessee 27 Troubleshooting Guide sssssssssseseesssssssseosrerssessssssssstesersssss
10. asional 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 3 2 mL Solution Ill Invert and rotate the tube gently until flocculent white precipitates form This may require a 2 3 minute incubation at room temperature with occasional mixing Note It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation If the mixture still appears viscous brownish or conglobated more mixing is required to completely neutralize the solution Centrifuge at 15 000 x g for 10 minutes at room temperature preferably at 4 C A compact white pellet will form 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 11 E Z N A Plasmid DNA Midi Kit Vacuum Protocol Optional Protocol for Column Eguilibration 11 12 13 14 T5 16 ve 18 19 20 12 Add 1 mL 3M NaOH to the HiBind DNA Midi Column Let sit at room temperature for 2 minutes Turn on the vacuum source to draw the NaOH through the column Turn off the vacuum Saat ic Transfer 3 5 mL cleared supernatant from Step 8 by CAREFULLY
11. aspirating it into the HiBind DNA Midi Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Midi Column Turn on the vacuum source to draw the supernatant through the column Turn off the vacuum Repeat Steps 11 13 until all of the cleared supernatant has been transferred to the HiBind DNA Midi Column Add 3 mL HBC Buffer Note HBC Buffer must be diluted with isopropanol 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 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 21 22 23 24 25 26 27 28 E Z N A Plasmid DNA Midi Kit Vacuum Protocol Repeat Steps 18 20 for a second DNA Wash Buffer wash step Transfer the HiBind DNA Midi Column to a 15 mL Collection Tube provided Centrifuge the empty HiBind DNA Midi Column at 4 000 x g for 2 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 not prov
12. ce of purified DNA does not accurately reflect quality of the plasmid DNA A A 99 ratio is too high or too low Check the absorbance of the ethanol between DNA Wash Buffer is diluted 250 nm and 300 nm Do not use ethanol with high with ethanol containing absorbance impurities Trace impurities may remain on the column after washing and can contribute to the absorbance Confirm that the RNase A was added to Solution 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 Plasmid DNA is contaminated with RNA RNase A treatment is insufficient Background reading is Spin the DNA sample at maximum speed for 1 min high due to fine silica ute use the supernatant to repeat the absorbance particulates readings Purification is incomplete foals p f Reduce the initial volume of culture due to column overloading Do not use cultures that have grown for more than 24 hours or are in the cell death phase nated with chromosomal F gt DNA Do not vortex or vigorously shake the cells during the lysis reaction or neutralization 4 000 x g centrifuge not available 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
13. cket rotor at 4 000 x g for liquids to pass insufficient g force through efficiently Reduce the lysis time Solution II to 3 minutes Alkaline lysis is prolonged or until the suspended cells form a clear viscous solution Confirm the cell density by measuring OD To Too many or too few cells calculate the volume of culture to use take the were used desired cell mass 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 instructions diluted with ethanol on Page 6 HBC Buffer not diluted with Prepare HBC Buffer according to instructions on Page isopropanol 6 High molecular weight DNA contamination of product Cell lysate over mixed upon Do not vortex or mix aggressively after adding addition of Solution II Solution Il e Overgrown culture contains lysed cells and degraded Culture overgrown DNA Do not grow cell for longer than 16 hours 28 Troubleshooting Guide RNA visible on agarose gel Check that RNase A provided with the kit has been used If Solution is more than 6 months old add more RNase A RNase A not added to Solution DNA floats out of well while loading agarose gel Ethanol has not been Centrifuge column as instructed to dry the column removed completely from before elution column following wash Incubate columns for 10 minutes at 65 C to com steps pletely dry membrane after centrifugation step Absorban
14. e 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 215 000 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 26 Low Copy Number Plasmids and Cosmids E Z N A 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 bacteria use the following modified protocol Note The E Z N A Plasmid DNA Midi Kit and the E Z N A 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 30 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 Plasmid DNA Midi Kit and 200 400 mL bacterial culture for the E Z N A Plasmid DNA Maxi Kit 2 Pellet the bacterial cells by centrifugation 3 Decant or aspirate and discard the cultur
15. e media 4 Perform Steps 4 8 in the standard protocols with double volumes of Solution Solution Il and Solution III 5 Continue with Step 9 of the standard protocols by following the wash drying and elution steps There is no need to increase the volumes of HBC Buffer DNA Wash Buffer or Elution Buffer 27 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 Poor Cell Lysis 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 Do not incubate cultures for more than 16 hours at Bacterial culture is 37 C overgrown or not fresh Storage of cultures for extended periods prior to plasmid isolation is detrimental Low elution efficiency Sterile deionized water must be 8 5 pH Such plasmids may yield as little as 0 1 ug plasmid Low copy number plasmid DNA from a1 mL overnight culture used Double culture volume and follow the Low Copy Number Plasmid and Cosmid DNA Protocol Columns were spun ina For the Midi and Maxi kits the columns must be spun fixed angle rotor or with in a swing bu
16. 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 E Z N A Plasmid DNA Midi Kit Vacuum Protocol E Z N A Plasmid DNA Midi Kit Vacuum Protocol All centrifugation steps after Step 8 should be performed with a swing bucket rotor for maximum plasmid DNA yields All centrifugation steps should be carried out at room temperature unless otherwise noted If high yields of low copy number plasmid DNA are desired see Low Copy Number Plasmids and Cosmids on Page 27 Materials and Equipment to be Supplied by User 100 ethanol do not use denatured alcohol lsopropanol Centrifuge with swing bucket rotor capable of 4 000 x g Centrifuge capable of 15 000 x g Nuclease free 15 mL and 50 mL centrifuge tubes 30 mL or 50 mL centrifuge tubes capable of withstanding 15 000 x g Vacuum manifold Optional Water bath incubator or heat block capable of 65 C Optional Sterile deionized water Optional 3M NaOH Before starting 10 Check Solution Il and Solution Ill for precipitation before use Redissolve any precipitates by warming to 37 C Prepare DNA Wash Buffer and Solution 1 according to Preparing Reagents section on Page 6 Optional Heat Elution Buffer to 65 C if plasmid DNA is gt 10 kb Transfer 20 50 mL overnight culture to a 50 mL centrifuge tube not provided Note The o
17. ep 1 Optional Water bath incubator or heat block capable of 65 C Optional Sterile deionized water Optional 3M NaOH Before starting 18 Check Solution Il and Solution Ill for precipitation before use Redissolve any precipitates by warming to 37 C Prepare DNA Wash Buffer and Solution 1 according to Preparing Reagents section on Page 6 Optional Heat Elution Buffer to 65 C if plasmid DNA is gt 10 kb 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 cell 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 27 10 E Z N A 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 wal
18. ided Add 0 5 1 mL Elution Buffer or sterile deionized water 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 13 E Z N A Plasmid DNA Midi Kit Centrifugation Protocol E Z N A Plasmid DNA Midi Kit Centrifugation Protocol All centrifugation steps after Step 8 should be performed with a swing bucket rotor for maximum plasmid DNA yields All centrifugation steps should be carried out at room temperature unless otherwise noted If high yields of low copy number plasmid DNA are desired see Low Copy Number Plasmids and Cosmids on Page 27 Materials and Equipment to be Supplied by User 100 Ethanol do not use denatured alcohol lsopropanol Centrifuge with swinging bucket rotor capable of 4 000 x g Centrifuge capable of 15 000 x g Nuclease free 15 mL and 50 mL centrifuge tubes 30 mL or 50 mL centrifuge tubes capable of withstanding 15 000 x g Optional Water bath incubator or heat block capable of 65 C Optional Sterile deionized water Optional 3M NaOH Before starting 14 Check Solution Il and Solution Ill for precipitation before use Redissolve any
19. iltrate and reuse the collection tube E Z N A Plasmid DNA Maxi Kit Centrifugation Protocol 20 21 22 23 24 25 26 Repeat Steps 17 19 for a second DNA Wash Buffer wash step 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 not provided Add 1 5 3 mL Elution Buffer or sterile deionized water directly to the center of the column membrane 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 25 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 volum
20. l of the bottle Add 12 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 Transfer the cell suspension to a 30 mL or 50 mL centrifuge tubes capable of withstanding 15 000 x g not provided Add 12 mL Solution Il Invert and rotate the tube gently 10 12 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 Add 16 mL Solution Ill Invert and rotate the tube gently until flocculent white precipitates form This may require a 2 3 minute incubation at room temperature with occasional mixing Note It is vital that the solution is mixed thoroughly and immediately after the addition of Solution Ill to avoid localized precipitation Centrifuge at 15 000 x g for 10 minutes at room temperature preferably at 4 C A compact white pellet will form Promptly proceed to the next step 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
21. ng D Vacuum Source review tables below for pressure settings Manifold Recommended Pressure mbar VAC 08 200 to 600 Conversion from millibars Multiply by Millimeters of mercury mm Hg 0 75 Inches of mercury inch Hg 0 0295 Tors Tort Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Illustrated Vacuum Setup Bge a Heo 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 Plasmid DNA Kits 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 activities 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 II and Solution Ill if problems are encountered with strains such as TG1 and Top10F Inoculation Bacterial cul
22. on on Page 6 5 Transfer the cell suspension to a 30 mL or 50 mL centrifuge tubes capable of withstanding 15 000 x g not provided 6 Add 2 25 mL Solution Il 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 7 Add 3 2 mL Solution Ill Invert and rotate the tube gently until flocculent white precipitates form This may require a 2 3 minute incubation at room temperature with occasional mixing Note It is vital that the solution is mixed thoroughly and immediately after the addition of Solution Ill to avoid localized precipitation 8 Centrifuge at 15 000 x g for 10 minutes at room temperature preferably at 4 C A compact white pellet will form Promptly proceed to the next step Note Steps 9 26 should be performed in a swing bucket rotor for maximum plasmid DNA yield All of centrifugation steps should be carried out at room temperature 9 Insert a HiBind DNA Midi Column into a 15 mL Collection Tube supplied 15 E Z N A Plasmid DNA Midi Kit Centrifugation Protocol Optional Protocol for Column Eguilibration 10 11 12 13 14 15 16 ve 18
23. ptimal 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 27 Centrifuge at 4 000 x g for 10 minutes at room temperature 10 E Z N A 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 25 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 Transfer the cell suspension to a 30 mL or 50 mL centrifuge tubes capable of withstanding 15 000 x g not provided Add 2 25 mL Solution Il 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 occ
24. re that all traces of the medium are removed use a clean paper towel to blot excess liquid from the wall of the tube 4 Add 12 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 5 Transfer the cell suspension to a 30 mL or 50 mL centrifuge tubes capable of withstanding 15 000 x g not provided 6 Add 12 mL Solution Il Invert and rotate the tube gently 10 12 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 7 Add 16 mL Solution Ill Invert and rotate the tube gently until flocculent white precipitates form This may require a 2 3 minute incubation at room temperature with occasional mixing Note It is vital that the solution is mixed thoroughly and immediately after the addition of Solution Ill to avoid localized precipitation 8 Centrifuge at 15 000 x g for 10 minutes at room temperature preferably at 4 C A compact white pellet will form Promptly proceed to the next step Note Steps 9 26 should be performed with a swing bucket rotor for maximum plasmid
25. re using the E Z N A Plasmid DNA Midi Kit Up to 600 1200 ug high copy number plasmid DNA or 50 300 ug low copy number plasmid DNA can be purified from 50 200 mL overnight culture using E Z N A Plasmid DNA Maxi Kit New In this Edition HB Buffer has been replaced by HBC Buffer Isopropanol is required and supplied by the user e Equilibration Buffer is no longer included with this kit An optional Column Equilibration Protocol has been added to the protocol for your convenience Equilibration Buffer is replaced with 3M NaOH provided by the user Storage and Stability All of the E Z N A 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 tem perature Store Solution II tightly capped when not in use During shipment or storage in cool ambient conditions precipitates may form in HBC Buffer Solution II and Solution Ill Dissolve such deposits by warming the solution at 37 C and gently shaking 2 Yield and Ouality 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
26. ry 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 not supplied Add 0 5 1 mL Elution Buffer or sterile deionized water 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 17 E Z N A Plasmid DNA Maxi Kit Vacuum Protocol E Z N A Plasmid DNA Maxi Kit Vacuum Protocol All centrifugation steps after Step 8 should be performed in swing bucket rotor for maximum plasmid DNA yields All centrifugation steps should be carried out at room temperature unless otherwise noted If high yields of low copy number plasmid are desired see Low Copy Number Plasmids and Cosmids on Page 27 Materials and Equipment to be Supplied by User 100 ethanol do not use denatured alcohol lsopropanol Centrifuge with swing bucket rotor capable of 4 000 x g Centrifuge capable of 15 000 x g Vacuum manifold Nuclease free 50 mL centrifuge tubes 30 mL or 50 mL centrifuge tubes capable of withstanding 15 000 x g Appropriate centrifuge bottle for St
27. sssseseeee 28 PCL SPAT Gisserot UA KAMUA MAE NUU AINA 30 Manual Revision May 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction The E Z N A family of products is an innovative system that radically simplifies extraction and purification of nucleic acids from a variety of sources Key to the system is the Omega Bio tek s proprietary HiBind matrix that avidly but reversibly binds DNA or RNA under optimized conditions allowing proteins and other contaminants to be removed Nucleic acids are easily eluted with deionized water or low salt buffer The E Z N A Plasmid DNA Midi and Maxi Kits combine the power of HiBind technology with the time tested consistency of alkaline SDS lysis of bacterial cells to deliver high quality DNA HiBind DNA columns facilitate the binding washing and elution steps thus enabling multiple samples to be processed simultaneously Following lysis the DNA is bound to the silica membrane and contaminants are removed with a simple wash step Plasmid DNA is eluted with low salt buffer Plasmid DNA purified by this system is suitable for automated fluorescent DNA sequencing restriction endonuclease digestion transfection of mammalian cells and other manipulations Yields vary according to plasmid copy number E coli strain and growth conditions Up to 100 250 ug high copy number plasmid DNA or 10 50 ug low copy number plasmid DNA can be purified from 20 50 mL overnight cultu
28. the vacuum for 5 minutes to completely dry the HiBind 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 not provided Add 1 5 3 mL Elution Buffer or sterile deionized water 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 21 E Z N A Plasmid DNA Maxi Kit Centrifugation Protocol E Z N A Plasmid DNA Maxi Kit Centrifugation Protocol All centrifugation steps after Step 8 should be performed with a swing bucket rotor for maximum plasmid DNA yields All centrifugation steps should be carried out at room temperature unless otherwise noted If high yields of low copy number plasmid are desired see Low Copy Number Plasmids and Cosmids on Page 27 Materials and Equipment to be Supplied by User 100 ethanol do not use denatured alcohol lsopropanol Centrifuge with swing bucket rotor capable of 4 000 x g Centrifuge capable of 15 000 x g Nuclease free 50 mL centrifuge tubes 30 mL or 5
29. tures for plasmid 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 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
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