E-Z96 FastFilter Plasmid Kit - Omega Bio-Tek
Contents
1. Guideline for Vacuum Manifold Lysate Clearance Setup DNA Bind amp Wash Setup Lysate Clearance Plate E Z 96 DNA Plate Vacuum Manifold Collar Vacuum Manifold Collar E Z 96 DNA Plate Waste Collection 96 well Square well Plate Waste Collection SS Vacuum Manifold Base E Z 96 DNA Plate Vacuum Manifold Collar 96 well Microplate 500 uL OO l Waste Collection ay Vacuum Manifold Base E Z 96 FastFilter Plasmid DNA Kit Vacuum Protocol E Z 96 FastFilter Plasmid DNA Kit Vacuum Protocol All centrifugation steps used are performed at room temperature Materials and Equipment to be Supplied by User e Centrifuge with swing bucket rotor capable of 4 000 x g Rotor adapter for 96 well Square well Plates Standard vacuum manifold Omega Bio tek Cat VAC 03 recommended e Optional Vacuum oven or incubator capable of 70 C e Optional Sterile deionized water Before Starting e Prepare Solution DNA Wash Buffer and HBC Buffer according to Preparing Reagents section on Page 5 e Optional Set the oven or incubator to 70 C 1 Grow 1 0 1 5 mL E coli LB cultures in a 96 well Square well Plate 2 2 mL provided at 37 C with agitation 180 300 rpm for 20 24 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 Seal2. D1097 01 480 mL D1097 02 800 mL per bottle 3 Dilute HBC Buffer with isopropanol as follows and store at room temperature Kie Isopropanol to be Added D1097 02 109 mL per bottle 4 Check Solution Il and Solution Ill for precipitation before use Redissolve any precipitation by warming to 37 C Cleaning of 96 well Square well Plates The 96 well Square well Plates supplied with this kit are reusable To avoid cross contamination rinse the plates thoroughly with tap water after each use Soak the plates in 0 5M HCI for 5 minutes then wash thoroughly with distilled water The 96 well Square well Plates also can be autoclaved following washing Guideline for Vacuum Manifold The following is required for use with the Vacuum Protocol A Vacuum Manifold We recommend Omega Bio tek s VAC 03 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 Recommended Pressure mbar VAC 03 200 to 400 Conversion from millibars Multiply by Millimeters of Mercury mmHg 0 75 Kilopascals kPa 0 1 Inches of Mercury inchHg 0 0295 Tors Tor Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Illustrated Vacuum Setup a _S Omega Bio tek s VAC 03 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask
3. completely dry the HiBind matrix Traces of ethanol remain on column prior to elution Cell lysis process should not be over 5 minutes It may cause DNA to permanently denatured DNA is permanently denatured Ordering Information The following components are available for purchase separately Call Toll Free Number 1 800 832 8896 Solution 250 mL PS001 Solution II 250 mL PS002 Solution III 250 mL PS003 Elution Buffer 100 mL PDR048 DNA Wash Buffer 100 mL PS010 RNase A 400 uL AC117 EZ 96 DNA Plate 10 BD96 01 Sealing Film 100 box AC1200 01 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 19 Notes 20
4. 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 sometimes can be determined best 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 concatemers may also be present Spin Protocols Vacuum Protocol Resuspend Resuspend Lyse Lyse Neutralize Neutralize Clear lysate with Lysate Clearance Plate Clear lysate with Lysate Clearance Plate Vacuum Bind and Bind and Wash Twice Vacuum Dry Membrane O hm Kit Contents TS E semaisaurenetnueaamy 1 o e svermer 1 f e ewon o d Storage and Stability All of the Z 96 FastFilter Plasmid Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows Solution I once RNase A is added should be stored at 2 8 C All other materials should be stored at room temperature Solution II must be tightly capped when not in use Preparing Reagents 1 Add the vial of RNase A to the bottle of Solution and store at 2 8 C 2 Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature kie 100 Ethanol to be Added D1097 00 120 mL
5. 96 DNA Plate and vigorously tap the plate on a stack of paper towels until no liquid comes out Remove any residual moisture from the tips of the E Z 96 DNA Plate with a clean paper towel Optional Place the E Z 96 DNA Plate into a vacuum oven or incubator set at 70 C for 10 minutes to further dry the plate 15 E Z 96 FastFilter Plasmid DNA Kit Centrifugation Protocol 26 27 28 29 30 31 16 Place the E Z 96 DNA Plate onto a 96 well Microplate 500 uL provided Add 100 150 uL Elution Buffer 10 mM Tris HCl pH 8 5 or sterile deionized water to each well Let sit for 2 minutes at room temperature Centrifuge at 3 000 x g for 5 minutes Seal the 96 well Microplate with caps or sealing film not provided Store 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 800 832 8896 Possible Problems and Suggestions Only use LB or YT medium containing antibiotic s Do not use more than 2 mL culture with high copy plasmids Cells may not be dispersed adequately prior to addition of Solution Il Mix cell suspension Poor cell lysis to completely disperse Increase incubation time with Solution II to obtain a clear lysate Solution Il if not tightly closed may need to be replaced Bacterial culture Reduce quantity of starting material overgrown or not f
6. LB cultures in a 96 well Square well Plate 2 2 mL provided at 37 C with agitation 180 300 rpm for 20 24 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 3 Centrifuge at 1 500 2 000 x g for 5 minutes at room temperature 4 Remove the sealing film and discard the supernatant 5 Dry the plate by placing upside down on a paper towel to remove excess media 13 E Z 96 FastFilter Plasmid DNA Kit Centrifugation Protocol Add 250 uL Solution I RNase A Pipet up and down to completely resuspend the cell pellet Note RNase A must be added to Solution prior to use Please see Page 5 for instructions Add 250 uL Solution Il Mix by gently shaking and rotating the plate for 1 minute to obtain a cleared lysate A 2 3 minute incubation at room temperature may be necessary Note Avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity Store Solution II tightly capped when not in use Add 350 uL Solution Ill Dry the top of the plate with a paper tower Seal the plate with sealing film Invert the plate gently 5 6 times until a flocculent white precipitate forms Optional Protocol for Plate Equilibration 10 11 12 13 14 14 Place a E Z 96 DNA Plate on to a 96 well deep well plate not provided Add 100 uL 3M NaOH to ea
7. the plate with sealing film 3 Centrifuge at 1 500 2 000 x g for 5 minutes at room temperature 4 Remove the sealing film and discard the supernatant 5 Dry the plate by placing upside down on a paper towel to remove excess media 6 Add 250 uL Solution I RNase A Pipet up and down to completely resuspend the cell pellet Note RNase A must be added to Solution prior to use Please see Page 5 for instructions E Z 96 FastFilter Plasmid DNA Kit Vacuum Protocol 7 Add 250 uL Solution II Mix by gently shaking and rotating the plate for 1 minute to obtain a cleared lysate A 2 3 minute incubation at room temperature may be necessary Note Avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity Store Solution II tightly capped when not in use 8 Add 350 uL Solution Ill Dry the top of the plate with a paper tower Seal the plate with sealing film Invert the plate gently 5 6 times until a flocculent white precipitate forms Optional Protocol for Plate Equilibration 1 AU RWN Prepare the vacuum manifold according to manufacturer s instructions For Omega s VAC 03 manifold set up the manifold as follows a Place the Waste Collection container inside the Vacuum Manifold Base b Place the Vacuum Manifold Collar squarely over the base c Place the E Z 96 DNA Plate over the Vacuum Manifold Collar d Seal the unused wells with sealing film Add 100 uL 3M NaOH to each well Turn on th
8. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z 96 FastFilter Plasmid Kit D1097 00 1 x 96 preps D1097 01 4x 96 preps D1097 02 20 x 96 preps May 2013 For research use only Not intended for diagnostic testing E Z 96 FastFilter Plasmid DNA Kit Table of Contents Introduction and Yield ssssssssssssssessessesosseseosteseoseeseeeeteereereeeseeeeeerreeeerereereseee 2 Illustrated Protocelsciceicencicnincnnnnincaamenneeaintoniainan 3 Kit Contents Storage and Stam iitysa cect ics ces cence 4 Preparing RedgeNt Sienra ernea eese ea Ee a T eE Ei 5 Guidelines for Vacuum Manifold sssssssssesssssssssssseosserssssssssseseeeseesssssssseeesees 6 E Z 96 FastFilter Plasmid DNA Vacuum Protocoll ccscssecsessssseeesseseeeees 8 E Z 96 FastFilter Plasmid DNA Centrifugation Protocol 10 Troubleshooting GUIAC ccecsesssessesssssssssecssscsessscssccsecsesssccsscsnsencesseseeaneesecsees 13 DIK E121 AS AITEN E EIA tasetssct tees aeatuautetueetieceess 15 Manual Revision May 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction The E Z 96 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 HiBind matrix that avidly but reversibly binds DNA or RNA under optimized conditions allowing proteins and other contaminants to be removed Nucl
9. ch well Centrifuge at 3 000 5 000 x g for 3 minutes Set the E Z 96 DNA Plate aside discard the filtrate and reuse the 96 well deep well plate PWN gt Place a E Z 96 Lysate Clearance Plate onto a 96 well deep well plate not provided Transfer all of the lysate from Step 8 to the E Z 96 Lysate Clearance Plate Let sit for 2 3 minutes at room temperature A white precipitate should float to the top Centrifuge at 3 000 x g for 5 minutes Discard the E Z 96 Lysate Clearance Plate Place the E Z 96 DNA Plate on top of the 96 well Square well Plate provided E Z 96 FastFilter Plasmid DNA Kit Centrifugation Protocol 13 16 17 18 19 20 21 22 23 24 25 Transfer the cleared cell lysate into the E Z 96 DNA Plate Centrifuge at 3 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Add 500 uL HBC Buffer to each well Note HBC Buffer must be diluted with isopropanol before use Please see Page 5 for instructions Centrifuge at 3 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Add 750 uL DNA Wash Buffer to each well Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 5 for instructions Centrifuge at 3 000 x g for 10 minutes Discard the filtrate and reuse the 96 well Square well Plate Repeat Steps 21 23 for a second DNA Wash Buffer wash step Remove the E Z
10. e vacuum source to draw the NaOH through the plate Turn off the vacuum Remove the sealing film Set the E Z 96 DNA Plate aside and disassemble the manifold 9 Set up the manifold for Lysate Clearance as follows refer to Page 7 for illustrations pang Place the Waste Collection container into the Vacuum Manifold Base Place the E Z 96 DNA Plate on top of the Waste Collection container Place the Vacuum Manifold Collar squarely over the base Place the E Z 96 Lysate Clearance Plate over the Vacuum Manifold Collar Seal the unused wells with sealing film 10 Immediately transfer the lysate from Step 9 to the E Z 96 Lysate Clearance Plate 11 Let sit for 5 minutes The white precipitate should float to the top E Z 96 FastFilter Plasmid DNA Kit Vacuum Protocol 12 13 14 T5 16 ve 18 19 20 21 22 23 24 10 Turn on the vacuum until all the lysate passes through the E Z 96 Lysate Clearance Plate Turn off the vacuum Discard the E Z 96 Lysate Clearance Plate Carefully transfer the E Z 96 DNA Plate containing the cleared lysate onto the vacuum manifold collar refer to Page 7 for illustrations Seal the unused wells of E Z 96 DNA Plate with sealing film Turn on the vacuum until all the lysate passes through the E Z 96 DNA Plate Turn off the vacuum Add 500 uL HBC Buffer to each well Note HBC Buffer must be diluted with isopropanol before use Please see Page 5 fo
11. eic acids are easily eluted with deionized water or low salt buffer The E Z 96 FastFilter Plasmid DNA Kit combines the power of HiBind technology with the time tested consistency of alkaline SDS lysis of bacterial cells to deliver high quality plasmid DNA By using the E Z 96 DNA Plate up to 96 samples can be simultaneously processed in less than 90 minutes The E Z 96 Lysate Clearance Plate obviates time consuming centrifugation for the clearing of bacterial alkaline lysates It also has an average DNA recovery rate 10 to 30 higher than the manual centrifugation method Although yields vary according to plasmid copy number E coli strain and growth conditions a 1 mL overnight culture in LB medium typically produces 10 15 ug high copy plasmid DNA New In this Edition HB Buffer has been replaced by HBC Buffer Isopropanol is required and supplied by the user Equilibration Buffer is no longer included with 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 e 2mL Collection Plates are now called 96 well Square well Plates This is a name change only there has been no change to the plastic ware e 500 uL Collection Plates are now called 96 well Microplates This is a name change only there has been no change to the plastic ware Yield and Quality of DNA Determine the absorbance of an appropriate dilution
12. r instructions Turn on the vacuum until all the HBC Buffer passes through the E Z 96 DNA Plate Turn off the vacuum Add 750 uL DNA Wash Buffer to each well Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 5 for instructions Turn on the vacuum until all the DNA Wash Buffer passes through the E Z 96 DNA Plate Turn off the vacuum E Z 96 FastFilter Plasmid DNA Kit Vacuum Protocol 25 Repeat Steps 22 24 for a second DNA Wash Buffer wash step 26 Centrifuge at 3 000 5 000 x g for 15 minutes to dry the plate Note It is important to dry the plate membrane before elution Residual ethanol may interfere with downstream applications 27 Remove the E Z 96 DNA Plate from the vacuum manifold Vigorously tap the plate on a stack of paper towels until no liquid comes out Remove any residual moisture from the tips of the E Z 96 DNA Plate with a clean paper towel Optional Place the E Z 96 DNA Plate into a vacuum oven or incubator set at 70 C for 10 minutes to further dry the plate 28 Return the E Z 96 DNA Plate back onto the vacuum manifold collar 29 Turnon the vacuum for an additional 5 minutes This step will remove any remaining ethanol from the membrane 30 Turn off the vacuum 31 Remove the E Z 96 DNA Plate and the vacuum manifold collar 32 Set up the manifold for Elution as follows refer to Page 7 for illustrations a Place the Waste Collection container into
13. resh Low copy number plasmids may yield as little as 0 1 ug DNA from a 1 mL overnight culture Increase culture volume to 3 mL DNA Wash Buffer not Prepare DNA Wash Buffer according to diluted with ethanol instructions on Page 5 HBC Buffer not diluted Prepare HBC Buffer according to instructions with isopropanol on Page 5 High molecular Low copy number plasmid used No DNA eluted Do not vortex or mix aggressively after weight DNA op adding Solution II Adequate mixing is eee lysate upon addition of e P contamination f obtained by simply inverting and rotating Solution Il of product the plate Over mixing of cell 17 Problem Optical densities do not agree with DNA yield on agarose gel Problem RNA visible on agarose gel Problem Plasmid DNA floats out of well while loading agarose gel Problem Plasmid DNA will not perform in downstream application 18 Troubleshooting Guide Trace contaminants eluted from column increase A Make sure to wash plate as instructed Alternatively rely on agarose gel ethidium bromide electrophoresis for quantization RNase A not added to Solution Add 1 vial of RNase to each bottle of Solution Ethanol not completely removed from column following wash steps The E Z 96 DNA Plate must be washed with 100 ethanol and dried before elution Ethanol precipitation may be required following elution Follow the optional drying step to
14. the Vacuum Manifold Base b Place a 96 well Microplate 500 uL provided on top of the Waste Collection container c Place the Vacuum Manifold Collar squarely over the base Place the E Z 96 DNA Plate over the Vacuum Manifold Collar e Seal the unused wells with sealing film a 33 Add 100 150 ul Elution Buffer 10 mM Tris HCI pH 8 5 or sterile deionized water to each well 34 Let sit for 2 minutes at room temperature 11 35 36 37 38 12 E Z 96 FastFilter Plasmid DNA Kit Vacuum Protocol Turn on the vacuum for 5 10 minutes to elute the DNA from the plate Turn off the vacuum Disassemble the vacuum manifold remove the 96 well Microplate containing the eluted DNA and seal with caps or sealing film not provided Store at 20 C E Z 96 FastFilter Plasmid DNA Kit Centrifugation Protocol E Z 96 FastFilter Plasmid DNA Kit Centrifugation Protocol All centrifugation steps used are performed at room temperature Materials and Equipment to be Supplied by User e Centrifuge with swing bucket rotor capable of 4 000 x g Rotor adapter for 96 well Square well Plates e 96 well deep well plates e Optional Vacuum oven or incubator capable of 70 C e Optional Sterile deionized water Before Starting Prepare Solution DNA Wash Buffer and HBC Buffer according to Preparing Reagents section on Page 5 e Optional Set the oven or incubator to 70 C 1 Grow 1 0 1 5 mL E coli
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