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1. If beading has occurred gently shake or vortex container until particles have been resuspended in solution 10 11 12 13 14 T5 16 17 18 19 20 10 Mag Bind Plasmid Maxi Protocol Let sit at room temperature for 10 minutes with gently shaking or inverting Note For low copy number plasmid insolation a 25 minute or overnight incubation can increase yields at room temperature Place the tube on the magnet separation device and remove the supernatant after Mag Bind Particles CND have completely migrated to the walls of the tube Remove the tube from the magnet separation device Add 25 mL SPM Wash Buffer Vortex or pipet up and down to resuspend the Mag Bind Particles CND Place the tube on the magnet separation device and remove the supernatant after Mag Bind Particles CND have completely migrated to the walls of the tube Repeat Steps 13 14 for a second SPM Wash Buffer wash step Air dry the Mag Bind Particles CND pellet for 10 minutes at room temperature If necessary remove any liquid drop from the tube with a pipettor Note After 1 minute check the tube and remove any residual ethanol Add 1 2 mL Elution Buffer or TE Buffer Vortex or pipet up and down to resuspend the Mag Bind Particles CND Incubate at 37 C for 10 minutes Place the tube on the magnet separation device Transfer the cleared supernatant containing the DNA to a new 2 mL microcentrifuge tube Store
2. absorbance in the final product Plasmid DNA is contaminated with RNA RNase A treatment is insufficient Confirm that the RNase A Solution was added to Solution prior to first use The RNase A Solution may degrade due to high temperatures gt 65EC or prolonged storage gt 6 months at room temperature Purification is incomplete due to Reduce the initial volume of culture column overloading 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 Neutralization procedure Plasmid DNA is contaminated with chromosomal DNA 14 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 Product Part Number Solution 250 mL PSOO1 Solution II 250 mL PS002 Neutralization Buffer 250 mL PS004 Elution Buffer 100 mL PDR048 RNase A 400 pL 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 license 15 Notes
3. pBluescript vectors ColE14 300 500 100 180 pg pGEM vectors 300 400 100 200 ug pBR322 and its derivatives pMB1 15 20 10 20 ug PACYC and its derivatives p15A 37540 5 10 ug pSC101 and its derivatives pSC101 5 ug pGEM pMB1 300 700 100 200 ug ColE14 ColE14 15 20 10 20 ug Kit Contents Mag Bind Plasmid Maxi Kit M1257 02 Preparations Lysate Clearance Syringe Mag Bind Particles CND 3 3 mL Solution 225 mL Solution II 225 mL PFC Binding Buffer 200 mL Neutralization Buffer 225 mL RNase A 1 2mL SPM Wash Buffer 2x 165 mL Elution Buffer 90 mL User Manual vi Storage and Stability All of the Mag Bind Plasmid Maxi Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows Solution once RNase A is added and Mag Bind Particles CND at 2 8 C All other materials at 22 25 C Store Solution II tightly capped when not in use Check Solution II Buffer for precipitation before use Redissolve any precipitation by warming to 37 C Preparing Reagents Add vial of RNase A to the bottle of Solution provided and store at 2 8 C e Dilute SPM Wash Buffer with 100 ethanol as follows and store at room temperature M1257 01 210 mL M1257 02 385 mL per bottle Bacterial Culture Recommendations 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 an
4. this will shear chromosomal DNA and lower plasmid purity Store Solution II tightly capped when not in use Add 10 mL ice cold Neutralization Buffer Mix gently and thoroughly by inverting and rotating the tube 10 times until flocculent white precipitates form This may require a 2 minute incubation at room temperature with occasional mixing Note The solution must be mixed thoroughly This is vital to obtaining good yields If the mixture still appears viscous brownish and conglobated more mixing is required to completely neutralize the solution Prepare a Lysate Clearance Syringe by removing the plunger and place the barrel ina tube rack to keep the syringe upright Immediately pour the lysate from Step 5 into the barrel of the Lysate Clearance Syringe Allow the cell lysate to sit for 2 minutes White precipitates should float to the top Hold the Lysate Clearance Syringe barrel over a new 50 mL centrifuge tube 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 Add 150 uL Mag Bind Particles CND and 9 mL PFC Binding Buffer Mix well by inverting the tube a few times Important The Mag Bind Particles CND will settle and bead together at the bottom of their container after several hours Please check container before use
5. 00 mL overnight culture using the Mag Bind Plasmid Maxi Kit Yields vary according to plasmid copy number E coli strain and the growth conditions 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 value 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 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 Mag Bind Plasmis Maxi Kit are indicated in the following table pUC vectors pMBI 500 700 150 250 ug
6. DNA at 20 C DNA Precipitation The concentration of the eluted plasmid varies with copy number host strain and growing conditions In some cases residual ethanol may also be present in the eluted plasmid DNA To adjust the DNA concentration following plasmid DNA elution or for the removal of ethanol residue one can perform the following isopropanol precipitation 1 Carefully transfer the eluted plasmid to a clean tube suitable for precipitation Add 1 10 volume of 3M NaAC pH 5 2 and 0 7 volumes isopropanol room temperature Vortex to mix and centrifuge at gt 15 000 x g for 20 minutes at 4 C Carefully decant the supernatant 2 Wash DNA pellet once with 1 2 mL 70 ethanol and centrifuge at gt 15 000 x g for 10 minutes at 4 C Carefully decant the supernatant without disturbing the pellet and air dry the pellet for 10 minutes 3 Resuspend DNA pellet in 200 500 uL Elution Buffer depending on desired concentration of final product 11 Low Copy Number Plasmid Cosmid DNA Purification 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 midi copy number plasmid 1 2 ug mL culture bacteria use the following modified protocol 1 Starting bacterial volume Double the volume of starting culture from that of high copy number plasmids Use up to 200 400 mL bacterial culture for Maxi Preps Pellet the bacterial cel
7. Mag Bind Plasmid Maxi Kit Table of Contents WANOCIUICTION reenen an 2 Yield and Quality of DNA scninsvarisincadanisanidatianuinaas 3 Kit CONTEN tSas aa 4 Preparing Reagents Storage and Stability 5 Bacterial Culture RecommendationS ssssssssssssssseresessereeeee 6 Mag Bind Plasmid Maxi ProtoCol ssesssssseserseessssssesssessse 8 Modified Protocol S sisonsnnnin innnan 11 Troubleshooting Guide sessessersersesesesesrrseesrrerrerssessrsssssess 13 Orderin nienia AA EAEAN E 15 Manual Revision June 2012 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction The Mag Bind Plasmid Maxi Kits combine the power of Mag Bind technology with the time tested consistency of alkaline SDS lysis of bacterial cells to deliver high quality DNA Mag Bind Particles CND facilitate the binding washing and elution steps thus enabling multiple samples to be simultaneously processed Mag Bind Particles CND can be used on automated liquid handlers such as Beckman Coulter s Biomek FX and Tecan Freedom Evo This system also include a special filter cartridge which replaces the centrifugation step following alkaline lysis Plasmid DNA purified by this system is suitable for automated fluorescent DNA sequencing restriction endonuclease digestion transfection of mammalian cells and other manipulations Up to 600 1200 ug high copy number plasmid or 50 300 ug of low copy number plasmid can be purified from 50 2
8. d C600 These host strains yield high quality DNA with the Mag Bind Plasmid Maxi 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 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 Il and Neutralization 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 It should then be incubated for 12 16 hours at 37 C with vigorous shaking 300rpm 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 Kits are specially designed for use with cultures grown i
9. ested modifications with low copy number plasmid protocol Bacterial Clone is overgrown or not fresh 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 Too many or too few calculate the volume of culture to use take the desired cells were used cell mass and divide by the absorbance of the overnight culture at 600 nm No DNA Eluted DNA Wash Buffer not Prepare SPM Concentrate according to diluted with ethanol instructions on page 6 High molecular weight DNA contamination of product Over mixing of cell lysate upon addition of Solution II Do not vortex or mix aggressively after adding Solution Il Overgrown culture contain 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 completely been removed from Mag Dry for 5 minutes at 37 C to completely remove netic Beads following wash steps Absorbance of purified DNA does not accurately reflect quality of the plasmid A A ratio is too high or too low Check the absorbance of the ethanol between 250nm SPM Buffer is and 300nm Do not use ethanol with high absorbance diluted with ethanol Traces of impurities may remain on the binding column containing impurities after washing and contribute to the
10. ls by centrifugation Note Additional buffers can be purchased separately See Page 15 for purchasing information on additional buffers 2 Perform alkaline lysis steps by using double volumes of Solution Solution Il Neutralization Buffer Additional buffer for Solution Solution Il Neutralization 3 Continue with each step of the standard protocol by following the wash drying and elution steps There is no need to increase the volumes of SPM Wash Buffer or Elution Buffer 12 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 800 832 8896 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 Poor cell lysis the addition of Solution II Make sure to vortex cell suspension to completely disperse Solution II if not tightly closed may need to be replaced Prepare as follows 0 2 N NaOH 1 SDS Do not incubate cultures for more than 16 hr at 37 C Storage of cultures for extended periods prior to plasmid isolation is detrimental Low elution If using Endotoxin free water for elution adjust the pH of efficiency water must be pH 8 0 Such plasmids may yield as little as 0 1ug of DNA froma Low copy number 1 mL overnight culture Double culture volume and follow plasmid used sugg
11. n 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 NOTE As culture ages DNA yield may begin to decrease due to cell death and lysis within the culture Bacterial Culture Recommendations 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 Mag Bind Plasmid Maxi Protocol Mag Bind Plasmid Maxi Kit Protocol Materials and Equipment to be Supplied by User 100 ethanol Do not use denatured alcohol Nuclease free 50 mL centrifuge tubes Nuclease free 2 mL microcentrifuge tubes Magnetic stand capable of using 50 mL tubes e Ice bucket Incubator capable of 37 C Before Starting e Pre chill Neutralization Buffer on ice Preheat Elution Buffer to 70 C if plasmid DNA is gt 10kb Prepare SPM Wash Buffer and Solution according to the Preparing Reagents sec
12. tion on Page 5 Preheat an incubator to 37 C 1 Harvest cells Pellet 50 200 mL overnight culture by centrifugation Optimal volume to use depends on the culture density and plasmid copy number see instruction in the notes below Transfer appropriate volume of culture to a centrifuge tube and centrifuge at 4 000 x g for 10 minutes Note The optimal cell mass OD x mL culture is around 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 inefficient Furthermore the excessive viscosity of the lysate will require vigorous mixing which may result in shearing of genomic DNA and contamination of the plasmid DNA For low copy number plasmids see the instructions for Low Copy Number Plasmids on Page 12 2 Decant or aspirate medium To ensure that all traces of the medium are removed use a clean paper towel to blot excess liquid from the wall of the vessel 3 Add 10 mL Solution I RNase A to the bacterial pellet Resuspend cells completely by vortexing or pipetting up and down Note Complete resuspension of the cell pellet is vital for obtaining good yields Mag Bind Plasmid Maxi Protocol Add 10 mL Solution II Mix gently and thoroughly by inverting and rotating the tube 10 times to obtain a cleared lysate This may require a 2 minute incubation at room temperature with occasional mixing Note Avoid vigorous mixing as
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