E.Z.N.A.®Endo-free Plasmid DNA Giga Kit - Omega Bio-Tek
Contents
1. Preparing Reagents section on Page 7 E Z N A Endo free Plasmid DNA Giga Kit Protocol Inoculate 2 LLB ampicillin 50 ug mL medium placed in a 5 liter culture flask with E coli carrying desired plasmid and grow at 37 C with agitation for 12 16 hours 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 and JM109 Note Optimal growth conditions of bacteria is vital of obtaining maximal plasmid DNA yields The best conditions are achieved by picking a single colony from a freshly transformed or freshly grown plate to inoculate a 2 5 mL starter culture containing the appropriate antibiotic Incubate for 8 hours at 37 C with vigorous shaking 300 rpm Using a flask or vessel with a volume of a least 3 4 times the volume of the culture dilute the starter culture 1 500 to 1 1000 times into warm culture media containing antibiotics Grow at 37 C for 12 16 hr with vigorous shaking 300 rpm Following overnight bacterial growth an OD of 1 5 2 0 indicates a well grown culture For the best result determination of OD for each culture is recommended It is important to dilute the bacterial culture 10 to 20 fold to enable photometric measurement in the linear range between 0 1 and 0 5 OD We recommend a bacterial density of between 2 0 and 3 0 at OD When using nutrient rich media care should be taken ensure that the cell density does not2. The culture volume should not exceed 1 4 the volume of the container Culture Media The E Z N A Endo free Plasmid DNA Giga 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 Giga Column Note As culture ages DNA yield may begin to decrease due to cell death and lysis within the culture Important Notes on Bacterial Cultures 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 media 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 Preparing Reagents Add vial of RNase A to the bottle of Solution provided and store at 2 8 C Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature Kit 100 Ethanol to be Added D6234 00 200 mL D6234 01 200 mL per bottle Prepare ETR Binding Beads as follows 1
3. incubate cultures for more than 24 hr at 37 C overgrown or not Storage of cultures for extended periods prior to plasmid fresh isolation is detrimental Low elution efficiency If using Endotoxin free Water for elution adjust the pH of the water to pH 8 0 Alkaline lysis was Reduce the lysis time Solution II to 2 minutes or until the prolonged suspended cells form a clear viscous solution Too many or too few Confirm the cell density by measuring OD To calculate the cells were used volume of culture to use take the desired cell mass and divide by the absorbance of the overnight culture at 600 nm DNA Wash Buffer not Prepare DNA Wash Buffer according to the instructions on Page diluted with ethanol 7 High molecular weight DNA contamination of product Over mixing of cell lysate upon addition Do not vortex or mix aggressively after adding Solution Il of Solution Il Overgrown culture contains lysed cells and degraded Culture overgrown DNA Do not grow cell longer than 16 hours 13 Troubleshooting Guide Plasmid DNA floats out of well while loading agarose gel Ethanol has not been removed completely from column following wash steps Dry the column as instructed before elution Incubate columns for 10 minutes at 65 C to completely dry membrane after vacuum step for drying Absorbance of purified DNA does not accurately reflect quantity of the plasmid A A ratio is high or low Check the absorbanc
4. 5 Notes
5. Aliquot the required amount of ETR Binding Beads Each prep will require 15 mL ETR Binding Beads Prepare within 4 hours of processing 2 Centrifuge at 3 000 x g for 3 minutes 3 Aspirate and discard the supernatant 4 Add 2 volumes water not provided 5 Vortex to resuspend the ETR Binding Beads 6 Repeat Steps 2 5 for a second water wash 7 Centrifuge at 3 000 x g for 3 minutes 8 Aspirate and discard the supernatant 9 Add 1 volume water not provided 10 Vortex to resuspend the ETR Binding Beads E Z N A Endo free Plasmid DNA Giga Kit Protocol E Z N A Plasmid DNA Giga Kit Vacuum Protocol This Protocol is designed to isolate 10 mg of high Copy Number plasmids or 1 mg of low copy Number Plasmids from 1 L overnight cultures using the E Z N A Endo Free Plasmid Giga Kit Binding more than 10 mg of Plasmid DNA to the HiBind DNA Giga Column can increase processing time and cause the column to clog Materials and Equipment to be Supplied by User e 100 ethanol e Centrifuge with swing bucket rotor capable of 5 000 x g with adapters for 50 mL centrifuge tubes and 500 mL centrifuge bottles Vacuum pump capable of generating 200 to 600 mbar Vacuum manifold Cat No VAC 08 e 50mL centrifuge tubes 500 mL centrifuge bottles i e Nalgene 3120 Vortexer Ice bucket Before Starting Chill Neutralization Buffer on ice Prepare ETR Binding Beads DNA Wash Buffer and Solution according to the
6. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z N A Endo free Plasmid DNA Giga Kit D6234 00 2 preps D6234 01 5 preps August 2013 For research use only Not intended for diagnostic testing E Z N A Endo free Plasmid DNA Giga Kit Table of Contents INTPOCUCTION Lee cecesssccsescssscccsescsssccsceccscsesscecesescscsesecesececececececees Kit Contents and StOLAGE seccsecssecssecseecseceseceseecseccseerseeseeeees Guidelines for Vacuum Manifold sssecssccssecssecneeceeseees Important Notes on Bacterial CUItUES cesesssecceeeseeeees Preparing REAGENTS sesiscisisscassisossseesssascossseciveniasidccsvvaccevaessvieores Plasmid DNA Giga Kit Protocol esessssesssecssecseecseerseesseeses Troubleshooting GUIE csesssessessssssseseestecsecsessncsneeseeseesneesees OrdEriN Grisarna t E r Rre Manual Revision August 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 Endo Free Plasmid DNA Giga Kit combines time t
7. e of the ethanol between 250 nm and 300 nm Do not use ethanol with high absorbance Traces of impurities may remain on the binding column after washing and contribute to the absorbance in the final product DNA Wash Buffer is diluted with ethanol containing impurities Plasmid DNA is contaminated with RNA RNase A treatment is insufficient Background reading is Centrifuge the DNA sample at maximum speed for 1 minute high due to silica fines use the supernatant to repeat the absorbance readings Purification is incomplete due to Reduce the initial volume of culture column overloading Confirm that 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 Do not use cultures that have grown for more than 24 hours or contaminated with are in the cell death phase Do not vortex or vigorously shake chromosomal DNA the cells during the lysis reaction 14 Ordering Information The following components are available for purchase separately Call Toll Free at 800 832 8896 Vacuum Manifold VAC 08 Solution 250 mL PS001 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 1
8. e to apply the vacuum to the HiBind DNA Giga Column for 15 minutes Note It is important to dry the HiBind DNA Giga Column matrix before elution Residual ethanol may interfere with downstream applications Turn off the vacuum Remove the HiBind DNA Giga Column from the vacuum manifold Place the tip cap removed in Step 16 firmly on the HiBind DNA Giga Column Add 10 15 mL Endotoxin free Water directly to the center of the column matrix Let sit for 5 minutes at room temperature Remove the tip cap and hold the HiBind DNA Giga Column over a 50 mL centrifuge tube not provided Gently insert the plunger to expel the Endotoxin free Water into the 50 mL centrifuge tube Repeat Steps 30 33 for a second elution step Note To maintain a higher concentration of plasmid DNA the first eluate can be reused Store DNA at 20 C 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 Poor cell lysis 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 Il Make sure to vortex cell suspension to completely disperse bacterial cells Solution II if not tightly closed may need to be replaced Bacterial colony is Do not
9. ested consistency of alkaline SDS lysis of bacterial cells with Omega Bio tek s innovative high efficiency DNA binding technology to recover large scale high quality plasmid DNA This method facilitates the binding washing and elution steps thus enabling multiple samples to be simultaneously processed This kit uses a syringe format system that is designed to work in combination with the centrifugation steps following the alkaline lysis of bacterial cells The Lysate Clearance Filter Syringe completely removes SDS precipitates and clears bacterial lysates in a fraction of the time required by centrifugation alone Yields vary according to plasmid copy number E coli strain and growth conditions but 1L overnight culture in LB medium typically produces 5 10 mg high copy plasmid DNA Up to 1 liter overnight culture may be processed when working with low copy number plasmids The product is suitable for automated fluorescent DNA sequencing restriction endonuclease digestion transfection of mammalian cells and other manipulations New In this Edition The latest edition of this manual has been redesigned to enhance readability and protocol quality Kit Contents Product Number D6234 00 D6234 01 Preparations 2 2 HiBind DNA Giga Columns Solution 270 mL 3x 230mL Solution Il 270 mL 3x 230 mL Neutralization Buffer 270 mL PFC Binding Buffer 270 mL EWR Wash Buffer 90 mL ETR Binding Beads 35 mL DNA Wash Buffer 50 mL End
10. exceed an OD of 3 0 If using a frozen glycerol stock as the inoculum streak it onto an agar plate containing the appropriate antibiotic for single colony isolation Then pick a single colony and inoculate the 2 5 mL starter culture as described above Centrifuge at 5 000 x g for 10 minutes at room temperature to pellet bacteria Decant or aspirate the media and discard To ensure that all traces of media are removed use a clean paper towel to blot excess liquid from the wall of the vessel Add 125 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 7 Add 125 mL Solution Il Invert and rotate the tube gently 10 times to obtain a cleared lysate Let sit at room temperature for 3 minutes 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 10 11 12 13 10 E Z N A Endo free Plasmid DNA Giga Kit Protocol Add 125 mL ice cold Neutralization 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 so
11. lution 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 Centrifuge at 4 000 x g for 20 minutes Note Increasing centrifugation speed is helpful to completely remove the precipitated bacterial cell debris After centrifugation a tightly packed pellet indicates efficient lysis Transfer the supernatant into a new 500 mL centrifuge bottle Note After the centrifugation the supernatant should appear clear If any precipitates are present in the supernatant pass the sample through a filter paper suchas Miracloth or a coffee filter before continuing Add 15 mL ETR Binding Beads Vortex for 10 20 seconds to mix thoroughly Note ETR Binding Beads must be prepared before use Please see the instructions in the Preparing Reagents section on Page 7 Let sit for 5 minutes at room temperature Centrifuge at 4 000 x g for 10 minutes Carefully transfer the cleared supernatant to a new 500 mL centrifuge bottle Avoid transferring the ETR Binding Bead pellet Note The ETR Binding Beads contains high levels of endotoxins and should not be transferred to the new vessel Measure the volume of the supernatant 14 15 16 17 18 19 20 21 22 23 E Z N A Endo free Plasmid DNA Giga Kit Protocol Add 1 3 volume PFC Binding Buffer Vortex to mix thoroughly Prepare
12. n 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 Giga 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 Neutralization Buffer if problems are encountered with strains such as TG1 and Top10F Inoculation Bacterial cultures 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
13. otoxin free Water 50 mL RNase A TmL User Manual vV Storage and Stability All of the E Z N A Endo free Plasmid DNA Giga Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows Store ETR Binding Beads at 2 8 C Store RNase A at 2 8 C Store Solution I RNase A at 2 8 C after being combined see Page 7 All remaining components should be stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in Solution Il and PFC Binding Buffer Dissolve such deposits by warming the solution at 50 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 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 Conversion from millibars Multiply by Millimeters of mercury mmHg 0 75 Inches of mercury inch Hg Tors Tor Atmospheres atmos Pounds per Square Inch psi Vacuum Setup Omega Bio tek s VAC 08 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask Important Notes on Bacterial Cultures Growth and Culture of Bacteria Bacterial Strain Selectio
14. the vacuum manifold by following the manufacturer s instructions Remove and save the plunger and the tip cap from the HiBind DNA Giga Column Note DO NOT discard the plunger or tip cap They will be required later Connect the HiBind DNA Giga Column to the vacuum manifold Refer to the Illustrated Vacuum Setup on Page 4 for details Transfer the sample from Step 14 by CAREFULLY decanting it into the HiBind DNA Giga Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Giga Column Note Do not allow the HiBind DNA Giga Column to become empty until you have transferred all of the sample from Step 14 Leaving 1 mL solution in the LJ column during Step 18 reduces f gt foaming and clogging which decreases total processing time U and improves efficiency Add 40 mL EWR Wash Buffer to the HiBind DNA Giga Column Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Add 50 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 7 Turn on the vacuum source to draw the buffer through the column 11 24 25 26 27 28 29 30 31 32 33 34 35 12 E Z N A Endo free Plasmid DNA Giga Kit Protocol Turn off the vacuum Repeat Steps 22 24 for a second DNA Wash Buffer wash step Continu
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