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Mag-Bind® Plasmid DNA 96 Kit - Omega Bio-Tek

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1. Omega Bio tek s VAC 03 Other Compatible Vacuum Manifolds Qiagen OlAvac24 Sigma Aldrich VM20 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 03 200 to 600 Conversion from millibars Multiply by millimeters of mercury mmHg 0 75 inches of mercury inHg Tor Tom atmospheres atm pounds per square inch psi Illustrated Vacuum Setup Omega Bio tek s VAC 03 C Vacuum Tubing A Vacuum Manifold D Vacuum Source B Vacuum Flask Guidelines for Vacuum Manifold Lysate Clearance Setup with 1 2 mL 96 well Round well Plate E Z 96 Lysate Clearance Plate Vacuum Manifold Top 96 well Round well Plate 1 2 mL y Vacuum Manifold Bottom Magnetic Separation Devices MSD 01B Radial Magnet Radial magnet can be used with 1 2 mL or 2 0 mL 96 well deep well plates where the posts of the magnets can fit in between the wells The magnetic beads will form a line along the inside of the wells making it ideal for washing by plate shakers or for larger volume extractions Note Check the volumes used in the protocol and the speed of plate shaker to ensure no cross contamination will occur Alp Aqua 96R Magnetic Stand A001219 Ideal for customers looking to automate the extractions due to an integrated spring that allows for easy and c
2. Ca OMEGA Innovations in nucleic acid isolation Product Manual Mag Bind Plasmid DNA 96 Kit M1256 00 1 x 96 preps M1256 02 24 x 96 preps September 2013 For research use only Not intended for diagnostic testing Mag Bind Plasmid DNA 96 Kit Table of Contents Introduction ee nen Kit Contents Storage and Stability Preparing Reagentsaun au Guidelines for Vacuum Manifold esesssseecsseseesseessesseees Magnetic Separation DeVICES sssssssscsssseessecessessesssseseees Mag Bind Plasmid DNA 96 Protocol Troubleshooting Guides css Ordering Information usssssesesessenseenssnnssnnsenssenssenssennne Manual Revision September 2013 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction The Mag Bind 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 Omega Bio tek s proprietary Mag Bind Particles that avidly but reversibly binds DNA or RNA under certain optimal conditions allowing proteins and other contaminants to be removed Nucleic acids are easily eluted with deionized water or low salt buffer The Mag Bind Plasmid DNA 96 Kit combines the power of Mag Bind technology with the time tested consistency of alkaline SDS Iysis of bacterial cells to deliver high quality plasmid DNA By using a 96 well format up to 96 samples can be simultaneously processed in less tha
3. R Add 250 uL SPM Wash Buffer to each sample Note SPM Wash Buffer must be diluted with ethanol prior to use Please see Page 4 for instructions Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Place the plate on the magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Leave the plate on the magnetic separation device for 5 10 minutes to air dry the Mag Bind Particles CNR Remove any residual liquid with a pipettor Remove the plate from the magnetic separation device Add 50 100 uL Elution Buffer Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Incubate 2 minutes at room temperature Note Incubation at 60 C rather than at room temperature may give a modest increase in DNA yield Place the plate on the magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Transfer the cleared supernatant containing purified DNA to a clean 96 well microplate not provided Store DNA at 20 C 11 Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the technical support st
4. aff toll free at 1 800 832 8896 Possible Problems and Suggestions Do not use more than 1 ml with high copy plasmids Cells may not be dispersed adequately prior to addition of Solution I Vortex cell suspen Poor cell lysis sion to completely disperse Increase incubation time with Solution Il to obtain a clear lysate Low DNA yields Solution Il if not tightly closed may need to be replaced Low copy number plasmid Such plasmids may yield as little as 0 1 ug used DNA from a 1 ml overnight culture Insufficient EWR Wash Time EWR wash buffer must be used for a mini mum of 120 seconds Mag Bind Particles CNR Lysate and PFC buffer Insufficient Bind Time must be mixed and incubated for a minimum FE AA 10 minutes Problem Cause Solution SPM Wash Buffer is not Prepare SPM Wash Buffer as instructed on the No DNA eluted diluted with ethanol label High molecular weight DNA con tamination Do not vortex or aggressively mix after add ing Solution Il Simply inverting and rotating tube to cover walls with viscous lysate Over mixing of cell lysate upon addition of Solution Il Optical densities Make sure to wash Mag Bind pellet as do not agree with race contaminants eluted instructed Alternatively rely on agarose gel DNA yield on aga from column increase A260 ethidium bromide electrophoresis or dye rose gel based method for quantification RNA visible on RNase A not added to Add 1 vial of RNase to eac
5. g up and down 20 times Note For low copy number plasmid isolation a 25 minute or overnight incubation at room temperature may increase yields Place the plate on the magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Remove the plate from the magnetic separation device Add 250 uL PFW Buffer to each sample Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Incubate for 1 minute Place the plate on the magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CNR Add 250 uL EWR Wash Buffer to each sample Resuspend the Mag Bind Particles CNR by vortexing or pipetting up and down 20 times Incubate for 2 minutes Place the plate on the magnetic separation device to magnetize the Mag Bind Particles CNR Let sit at room temperature until the Mag Bind Particles CNR are completely cleared from solution 24 25 26 27 28 29 30 31 32 33 34 35 Mag Bind Plasmid DNA 96 Kit Protocol Aspirate and discard the supernatant Do not disturb the Mag Bind Particles CN
6. h bottle of Solution agarose gel Solution L DNA floats out of well while loading agarose gel Ethanol not completely Increase air dry time before elution step removed before elution DNA will not The DNA plate must be washed with absolute perform in Traces of ethanol remain on ethanol and dried before elution Ethanol downstream column prior to elution precipitation may be required following applications elution Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 A A 96 well Microplate 500 uL 5 pk 96 well Microplate 500 uL 25 pk 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 13 Notes Notes Notes
7. n 60 minutes The new E Z 96 Lysate Clearance Plate eliminates time consuming centrifugation for clearing bacterial alkaline Iysates It also has an average DNA recovery rate 10 30 higher than the manual centrifuge method Yields vary according to plasmid copy number E coli strain and conditions of growth A 1 mL overnight culture in LB medium typically yields 10 15 ug high copy plasmid DNA The purified plasmid DNA can be used directly for automated fluorescent DNA sequencing as well as for other standard molecular biology techniques including restriction enzyme digestion New in this Edition PFC Buffer replaces isopropanol for binding group of DNA to magnetic beads Kit Contents Product Number M1256 00 M1256 02 Purifications 1 x 96 preps 24 x 96 preps Mag Bind Particles CNR 50 mL 1 96 well Microplate 500 uL Storage and Stability All Mag Bind Plasmid DNA 96 Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows the mixture of Solution I RNase A and Mag Bind Particles CNR should be stored at 2 8 C all other materials at 22 25 C Preparing Reagents 1 Dilute SPM Wash Buffer with 100 ethanol as follows and store at room temperature M1256 02 400 mL per bottle 2 Add the vial of RNase A to the bottle of Solution Store at 2 8 C Guidelines for Vacuum Manifold The following is required for use with the Vacuum Protocol A Vacuum Manifold We recommend
8. omplete supernatant removal without create air suctions which can lead to cross contamination or sample loss The spring can accomodate up to 1 25 mm of flexibility on the Z axis Contains 96 individual N48 NdFeB rings for fast magnetization response times of the magnetic beads Works with both PCR plates microtiter plates and round bottom deep well plates SBS footprint is designed to work with multiple automated liquid handlers and gripper grooves allow for easy placement on and off the magnetic stand Compatible with Corning Costar 3795 Abgene AB1127 1 2 mL square well Abgene AB 0661 2 2 mL Square well Nunc 260251 1 0 mL round well Mag Bind Plasmid DNA 96 Kit Protocol Mag Bind Plasmid DNA 96 Kit Protocol Materials and Equipment to be Supplied by User e Centrifuge with swinging bucket rotor at room temperature capable of 4 000 x g Adapter for 96 well deep well plates Magnetic separation device Optional Incubator capable of 60 C Refrigrator capable of 2 8 C 100 Ethanol Multi channel pipettor and tips Sealing film Cat AC1200 01 Multi channel Reservoirs Cat AC1331 01 96 well microplates Cat EZ9603 01 02 1 0 2 2 mL 96 well round well plates capatible with magnetic stand e Optional Vacuum pump or vacuum aspirator capable of achieving a vacuum of 20 24 inHg for vacuum protocol for clearing the cell lysate Optional Standard vacuum manifold Cat VAC 03 for vacuum protocol fo
9. on Il tightly capped when not in use Add 250 uL chilled 4 C Neutralization Buffer Mix by gently shaking and rotating the plate for 1 minute until a flocculent white precipitate forms Choose one of the following methods for lysate clearance A Clear the cell lysates with centrifugation 1 Place the E Z 96 Lysate Clearance Plate on top of the 96 well Microplate a 1 0 2 2 mL 96 well round well plate not provided 2 Transfer the lysate from Step 8 to the E Z 96 Lysate Clearance Plate 3 Let sit for 1 minute The white precipitate should float to the top 4 Centrifuge at 2 000 x g for 5 minutes B Clear the cell lysates with vacuum manifold 1 Place the 1 2 mL 96 well round well plate not provided into the base of the vacuum manifold See Page 6 for setup guidelines 2 Place the E Z 96 Lysate Clearance Plate on top of the manifold 3 Switch on vacuum source to draw the lysate through the membrane Add 20 uL Mag Bind Particles CNR and 235 uL PFC Buffer to the cleared lysate Mix thoroughly by pipetting up and down 20 times or shaking Important The Mag Bind Particles CNR will settle and clump together at the bottom of the bottle during storage Vortex the Mag Bind Particles CNR thoroughly before use 9 11 12 13 14 15 16 17 18 19 20 21 22 23 10 Mag Bind Plasmid DNA 96 Kit Protocol Let sit for 12 minutes at room temperature Mix thoroughly by vortexing or pipettin
10. r clearing the cell lysate Before Starting Prepare SPM Wash Buffer and Solution I RNase A according to instructions on Page 4 Chill Neutralization Buffer to 4 C Vortex the Mag Bind Particles CNR thoroughly before use Optional Heat the Elution Buffer to 60 C Note If you choose to vortex the samples in during the protocol below make sure to seal the plate completely to avoid any loss of sample or cross contamination 1 Grow 1 0 1 5 mL E coli LB cultures in a 2 mL 96 well culture plate at 37 C with agitation with for 16 20 hours Note It is strongly recommended that an endA negative strain of E coli be used for routine plasmid isolation Examples of such strains include DH5a and JM109 2 Seal the plate with sealing film 10 Mag Bind Plasmid DNA 96 Kit Protocol Centrifuge at 3 000 x g for 10 minutes at room temperature Remove the sealing film Discard supernatant Dry the plate by placing upside down on a paper towel to remove excess media Add 250 uL Solution Vortex or pipet up and down to completely resuspend the cell pellet Note RNase A must be added to Solution prior to use Please see Page 4 for instructions Add 250 uL Solution Il Mix by gently shaking and rotating the plate for 1 minute to obtain a cleared lysate A 5 minute incubation at room temperature may be necessary Note Avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity Store Soluti

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