MicroElute® Cycle-Pure Kit MicroElute® Gel - Omega Bio-Tek
Contents
1. Let sit at room temperature for 1 minute 21 17 18 22 MicroElute DNA Clean Up Kit Vacuum Protocol Centrifuge at maximum speed for 1 minute Note This represents approximately 80 9096 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration 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 Low DNA Yields Add more CP Buffer as indicated For DNA fragments 200 bp in size add up to 6 volumes of CP Buffer pH of the sample Add 10 20 uL sodium acetate pH 5 2 mixture is too high to the sample and mix Not enough CP Buffer added to sample Clogged Column in Gel Extraction No DNA Eluted Optical densities do not agree with DNA yield on agarose gel DNA sample floats out of well while loading agarose gel Increase incubation time Increase Binding Buffer XP2 volume Incompletely dissolved gel SPW Wash Buffer or DNA Wash Buffer was not diluted with ethanol Prepare SPW Wash Buffer or DNA Wash Buffer as instructed on Page 4 Wash column as instructed Alternatively rely on agarose gel ethidium bromide electrophoresis for quantification Trace contaminants eluted from column increase A 260 Ethanol not removed completely from Centrifuge column as instructed to dry column follo2. Transfer the MicroElute DNA Mini Column into a 2 mL Collection Tube provided Centrifuge the empty MicroElute DNA Mini Column at maximum speed for 2 minutes to dry the column Note This step is critical for removal of trace ethanol that may interfere with downstream applications Transfer the MicroElute DNA Mini Column into a clean 1 5 mL microcentrifuge tube not provided 17 18 19 20 MicroElute Cycle Pure Kit Vacuum Protocol Add 10 20 uL Elution Buffer TE Buffer or sterile deionized water directly to the center of column matrix Let sit at room temperature for 2 minutes Centrifuge at maximum speed for 1 minute Note This represents approximately 80 9096 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration Store DNA at 20 C 11 MicroElute Gel Extraction Kit Centrifugation Protocol MicroElute Gel Extraction Kit Centrifugation Protocol Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g 10096 ethanol 1 5 mL microcentrifuge tubes Incubator capable of 55 C Vortexer Optional 3M NaOH Optional Sterile deionized water Before Starting Prepare SPW Wash Buffer according to Preparing Reagents section on Page 4 Perform agarose gel ethidium bromide electrophoresis to fractionate DNA fragments Any type or grade of agarose may be used However it is strongly recommended that fresh TAE buff
3. g for 30 seconds Discard the filtrate and reuse the collection tube Uv OIN C3 Transfer no more than 700 uL DNA agarose solution from Step 5 to the MicroElute DNA Mini Column Note Each MicroElute DNA Mini Column has a total capacity of 10 ug DNA If the expected yield is larger divide the sample into the appropriate number of columns Centrifuge at 10 000 x g for 1 minute at room temperature Discard the filtrate and reuse collection tube Repeat Steps 7 9 until all of the sample has been transferred to the column Add 300 uL Binding Buffer XP2 Centrifuge at maximum speed 213 000 x g for 30 seconds at room temperature Discard the filtrate and reuse collection tube Add 700 uL SPW Wash Buffer Note SPW Wash Buffer must be diluted with 10096 ethanol prior to use Please see Page 4 for instructions Centrifuge at maximum speed for 1 minute at room temperature 13 MicroElute Gel Extraction Kit Centrifugation Protocol 16 Discard the filtrate and reuse collection tube Optional Repeat Steps 14 16 for a second SPW Wash Buffer wash step Perform the second wash step for any salt sensitive downstream applications 17 Centrifuge the empty MicroElute DNA Mini Column for 2 minutes at maximum speed to dry the column matrix Note It is important to dry the MicroElute DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications 18 Transfer the MicroElute DNA Mini Column to a
4. the vacuum Add 700 uL SPW Wash Buffer Note SPW Wash Buffer must be diluted with 10096 ethanol prior to use Please see Page 4 for instructions Turn on the vacuum source to draw the sample through the column Turn off the vacuum 18 19 20 21 22 23 24 25 MicroElute Gel Extraction Kit Vacuum Protocol Repeat Steps 15 17 for a second DNA Wash Buffer wash step Transfer the MicroElute DNA Mini Column to a clean 1 5 mL microcentrifuge tube Centrifuge the empty MicroElute DNA Mini Column for 2 minutes at maximum speed to dry the column matrix Note It is important to dry the MicroElute DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the MicroElute DNA Mini Column to a clean 1 5 mL microcentrifuge tube Add 10 20 uL Elution Buffer or sterile deionized water directly to the center of the column membrane Note The efficiency of eluting DNA from the MicroElute DNA Mini Column is dependent on pH If eluting DNA with sterile deionized water make sure that the pH is around 8 5 Let sit at room temperature for 2 minutes Centrifuge at maximum speed for 1 minute Note This represents approximately 7096 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration Store DNA at 20 C 17 MicroElute DNA Clean Up Kit Centrifugation Protocol MicroElute DNA Clean Up Kit Centrifugation P
5. 93 02 100 mL Dilute SPW Wash Buffer with 10096 ethanol as follows and store at room temperature EU ES 10096 Ethanol to be Added Dilute SPW Wash Buffer with 10096 ethanol as follows and store at room temperature D6296 01 100 mL D6296 02 100 mL per bottle Storage and Stability All of the MicroElute Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature During shipment or storage in cool ambi ent conditions precipitates may form in CP Buffer Dissolve such deposits by warming the solution at 37 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 OlAvac24 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 Torts or Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Vacuum Setup E s kaso Omega Bio tek s VAC 08 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask MicroElute Cycle Pure Kit Centrifugation Protocol MicroElute Cycle Pure Kit Centrifugation Protocol Materials and Equipment to be Supplied by User Microcentrifuge capable of at l
6. bio tek 2 OMEGA Innovations in nucleic acid isolation Product Manual MicroElute Cycle Pure Kit D6293 00 5 preps D6293 01 50 preps D6293 02 200 preps MicroElute Gel Extraction Kit D6294 00 5 preps D6294 01 50 preps D6294 02 200 preps MicroElute DNA Clean Up Kit D6296 00 5 preps D6296 01 50 preps D6296 02 200 preps April 2013 For research use only Not intended for diagnostic testing MicroElute Cycle Pure Kit MicroElute Gel Extraction Kit MicroElute DNA Clean Up Kit Table of Contents Introduction and Overview esent 2 Kit CONTENTS ET 3 Preparing Reagents Storage and Stability 4 Guideline for Vacuum Manifold sss 5 MicroElute Cycle Pure Centrifugation Protocol 6 MicroElute Cycle Pure Vacuum Protocol 9 MicroElute Gel Extraction Centrifugation Protocol 12 MicroElute Gel Extraction Vacuum Protocol 15 MicroElute DNA Clean Up Centrifugation Protocol 18 MicroElute DNA Clean Up Vacuum Protocol 20 Troubleshooting Guide aerem ittis crimes te irte 23 is eld ac iSo E MID IUE IUE 24 Manual Revision April 2013 5 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The E Z N A family of products is an innovative system that radically simplifies the extraction and purification of nucleic acids from a variety of sources The key to this system is the HiBind matrix t
7. clean 1 5 mL microcentrifuge tube 19 Add 10 20 uL Elution Buffer or sterile deionized water directly to the center of the column membrane Note The efficiency of eluting DNA from the MicroElute DNA Mini Column is dependent on pH If eluting DNA with sterile deionized water make sure that the pH is around 8 5 20 Let sit at room temperature for 2 minutes 21 Centrifuge at maximum speed for 1 minute Note This represents approximately 7096 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration 22 Store DNA at 20 C 14 MicroElute Gel Extraction Kit Vacuum Protocol MicroElute Gel Extraction Kit Vacuum Protocol Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g 10096 ethanol 1 5 mL microcentrifuge tubes Incubator capable of 55 C Vacuum Manifold Vortexer Optional 3M NaOH Optional Sterile deionized water Before Starting Prepare SPW Wash Buffer according to Preparing Reagents section on page 4 1 Perform agarose gel ethidium bromide electrophoresis to fractionate DNA fragments Any type or grade of agarose may be used However it is strongly recommended that fresh TAE buffer or TBE buffer be used as running buffer Do not reuse running buffer as its pH will increase and reduce yields 2 When adequate separation of bands has occurred carefully excise the DNA fragment of interest
8. ction Prepare the vacuum manifold according to manufacturer s instructions Connect the MicroElute DNA Mini Column to the vacuum manifold Optional Protocol for Column Equilibration 20 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column Switch on vacuum source to draw the buffer through the column Add 500 uL sterile deionized water to the MicroElute DNA Mini Column Switch on vacuum source to draw the water through the column Turn off the vacuum wi Pwn gt 10 11 12 13 14 15 16 MicroElute DNA Clean Up Kit Vacuum Protocol Transfer the sample from Step 3 to the MicroElute DNA Mini Column Turn on the vacuum source to draw the sample through the column Turn off the vacuum Add 700 uL SPW Wash Buffer Note SPW Wash Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 4 for instructions Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Repeat Steps 9 11 for a second SPW Wash Buffer wash step Centrifuge the empty MicroElute DNA Mini Column at maximum speed for 2 min utes to dry the column Note This step is critical for removal of trace ethanol that may interfere with downstream applications Transfer the MicroElute DNA Mini Column into a clean 1 5 mL microcentrifuge tube not provided Add 10 20 uL Elution Buffer TE Buffer or sterile deionized water directly to the center of column matrix
9. east 13 000 x g 10096 ethanol e 1 5 mL microcentrifuge tubes Vortexer e Optional 3M NaOH e Optional Sterile deionized water or TE Buffer Before Starting Prepare DNA Wash Buffer according to Preparing Reagents section on Page 4 1 Perform agarose gel ethidium bromide electrophoresis to analyze PCR product 2 Determine the volume of your PCR reaction 3 Transferthe sample into a clean 1 5 mL microcentrifuge tube 4 Add 5 volumes CP Buffer Note Volume refers to the size of your PCR reaction For example if your PCR reaction is 50 uL you would use 250 uL CP Buffer 5 Vortex to mix thoroughly Briefly centrifuge to collect any drops from the inside of the lid 6 Inserta MicroElute DNA Mini Column into a 2 mL Collection Tube provided MicroElute Cycle Pure Kit Centrifugation Protocol Optional Protocol for Column Equilibration 10 11 12 13 14 15 16 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column Centrifuge at 10 000 x g for 30 seconds Add 500 uL sterile deionized water to the MicroElute DNA Mini Column Centrifuge at 10 000 x g for 30 seconds Discard the filtrate and reuse the collection tube n sw Transfer the sample from Step 5 to the MicroElute DNA Mini Column Centrifuge at maximum speed 213 000 x g for 1 minute at room temperature Discard the filtrate and reuse collection tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with
10. er or TBE buffer be used as running buffer Do not reuse running buffer as its pH will increase and reduce yields When adequate separation of bands has occurred carefully excise the DNA fragment of interest using a wide clean sharp scalpel Minimize the size of the gel slice by removing extra agarose Determine the appropriate volume of the gel slice by weighing it in a clean 1 5 mL microcentrifuge tube Assuming a density of 1 g mL the volume of gel is derived as follows a gel slice of mass 0 3 g will have a volume of 0 3 mL Add 1 volume Binding Buffer XP2 Incubate at 60 C for 7 minutes or until the gel has completely melted Vortex or shake the tube every 2 3 minutes Important Monitor the pH of the Gel Binding Buffer mixture after the gel has completely 12 dissolved DNA yields will significantly decrease when the pH 8 0 If the color of the mixture becomes orange or red add 5 uL 5M sodium acetate pH 5 2 to bring the pH down After this adjustment the color of the Gel Binding Buffer mixture should be light yellow MicroElute Gel Extraction Kit Centrifugation Protocol 6 Insert a MicroElute DNA Mini Column in a 2 mL Collection Tube Optional Protocol for Column Equilibration 10 11 12 13 14 15 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column Centrifuge at 10 000 x g for 30 seconds Add 500 uL sterile deionized water to the MicroElute DNA Mini Column Centrifuge at 10 000 x
11. ethanol before use Please see the Preparing Reagents section on Page 4 for instructions Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse collection tube Repeat Steps 10 12 for a second DNA Wash Buffer wash step Centrifuge the empty MicroElute DNA Mini Column at maximum speed for 2 min utes to dry the column Note This step is critical for removal of trace ethanol that may interfere with downstream applications Transfer the MicroElute DNA Mini Column into a clean 1 5 mL microcentrifuge tube not provided Add 10 20 uL Elution Buffer TE Buffer or sterile deionized water directly to the center of column matrix MicroElute Cycle Pure Kit Centrifugation Protocol 17 Let sit at room temperature for 2 minutes 18 Centrifuge at maximum speed for 1 minute Note This represents approximately 80 9096 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration 19 Store DNA at 20 C MicroElute Cycle Pure Kit Vacuum Protocol MicroElute Cycle Pure Kit Vacuum Protocol Materials and Equipment to be Supplied by User Vacuum Manifold Microcentrifuge capable of at least 13 000 x g Nudlease free 1 5 mL microcentrifuge tubes 10096 ethanol e Optional 3M NaOH e Optional Sterile deionized water or TE Buffer Before Starting Prepare DNA Wash Buffer according to the Preparing Reagents section on Page 4 1 Perform agarose gel ethid
12. hat specifically 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 a low salt buffer The MicroElute Clean Up system designed for rapid DNA clean up includes e MicroElute Cycle Pure Kit for direct purification of double or single stranded PCR products 100 bp 10 kb from amplification reactions MicroElute Gel Extraction Kit for extraction of DNA fragments 70 bp 20 kb from standard or low melt agarose gels in TAE Tris acetate EDTA or TBE Tris borate EDTA buffer MicroElute DNA Clean Up Kit for general clean up of oligonucleotides and DNA up to 10 kb in size from enzymatic reactions e g labeling dephosphorylation restriction and tailing Binding Capacity Each MicroElute DNA Mini Column can bind 10 ug of DNA New in this Edition This manual has been edited for content and redesigned to enhance user readability 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 Kit Contents fawonsuner m sm om 7 Preparing Reagents Prepare the appropriate wash buffer as directed below depending on the kit ordered Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D62
13. ium bromide electrophoresis to analyze PCR product 2 Determine the volume of your PCR reaction 3 Transferthe sample into a clean 1 5 mL microcentrifuge tube 4 Add 5 volumes CP Buffer Note Volume refers to the size of your PCR reaction For example if your PCR reaction is 50 uL you would use 250 uL CP Buffer 5 Vortex to mix thoroughly Briefly centrifuge to collect any drops from the inside of the lid 6 Prepare the vacuum manifold according to manufacturer s instructions and connect the MicroElute DNA Mini Column to the manifold MicroElute Cycle Pure Kit Vacuum Protocol Optional Protocol for Column Equilibration 10 11 12 13 14 T5 16 10 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column Switch on vacuum source to draw the buffer through the column Add 500 uL sterile deionized water to the MicroElute DNA Mini Column Switch on vacuum source to draw the water through the column Turn off the vacuum Ur W N Transfer the entire sample from Step 5 to the MicroElute DNA Mini Column Switch on vacuum source to draw the sample through the column Turn off the vacuum Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 4 for instructions Switch on vacuum source to draw the DNA Wash Buffer through the column Turn off the vacuum Repeat Steps 10 12 for a second DNA Wash Buffer wash step
14. or 30 seconds Discard the filtrate and reuse collection tube Repeat Steps 8 10 for a second SPW Wash Buffer wash step Centrifuge the empty MicroElute DNA Mini Column at maximum speed for 2 min utes to dry the column Note This step is critical for removal of trace ethanol that may interfere with downstream applications Transfer the MicroElute DNA Mini Column into a clean 1 5 mL microcentrifuge tube not provided Add 10 20 uL Elution Buffer TE Buffer or sterile deionized water directly to the center of column matrix Let sit at room temperature for 1 minute Centrifuge at maximum speed for 1 minute Note This represents approximately 80 9096 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration Store DNA at 20 C 19 MicroElute DNA Clean Up Kit Vacuum Protocol MicroElute DNA Clean Up Kit Vacuum Protocol Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g Vacuum Manifold 10096 ethanol 1 5 mL microcentrifuge tubes Vortexer Optional 3M NaOH Optional Sterile deionized water or TE buffer Before Starting Prepare SPW Wash Buffer according to Preparing Reagents section on Page 4 Determine the volume of the enzymatic reaction Transfer the sample to a clean 1 5 mL microcentrifuge tube Add 3 volumes DP Buffer Vortex to mix thoroughly Note For example add 300 uL DP Buffer to 100 uL enzymatic rea
15. rotocol Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g 10096 ethanol 1 5 mL microcentrifuge tubes Vortexer Optional 3M NaOH Optional Sterile deionized water or TE buffer Before Starting Prepare SPW Wash Buffer according to Preparing Reagents section on Page 4 Determine the volume of the enzymatic reaction Transfer the sample to a clean 1 5 mL microcentrifuge tube Add 3 volumes DP Buffer Vortex to mix thoroughly Note For example add 300 uL DP Buffer to 100 uL enzymatic reaction Insert a MicroElute DNA Mini Column in a 2 mL Collection Tube Optional Protocol for Column Equilibration 18 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column Centrifuge at 10 000 x g for 30 seconds Add 500 uL sterile deionized water to the MicroElute DNA Mini Column Centrifuge at 10 000 x g for 30 seconds Discard the filtrate and reuse the collection tube Ui PwWwn gt Transfer the sample from Step 3 to the MicroElute DNA Mini Column Centrifuge at maximum speed 213 000 x g for 1 minute at room temperature MicroElute DNA Clean Up Kit Centrifugation Protocol 7 10 11 12 13 14 15 16 17 Discard the filtrate and reuse collection tube Add 700 uL SPW Wash Buffer Note SPW Wash Buffer must be diluted with ethanol before use Please see the Preparing Reagents section on Page 4 for instructions Centrifuge at maximum speed f
16. using a wide clean sharp scalpel Minimize the size of the gel slice by removing extra agarose 3 Determine the appropriate volume of the gel slice by weighing it in a clean 1 5 mL microcentrifuge tube Assuming a density of 1 g mL the volume of gel is derived as follows a gel slice of mass 0 3 g will have a volume of 0 3 mL 4 Add 1 volume Binding Buffer XP2 5 Incubate at 60 C for 7 minutes or until the gel has completely melted Vortex or shake the tube every 2 3 minutes 6 Prepare the vacuum manifold according to manufacturer s instructions T5 7 MicroElute Gel Extraction Kit Vacuum Protocol Connect the MicroElute DNA Mini Column to the vacuum manifold Optional Protocol for Column Equilibration 10 11 12 13 14 15 16 17 16 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column Switch on vacuum source to draw the buffer through the column Add 500 uL sterile deionized water to the MicroElute DNA Mini Column Switch on vacuum source to draw the water through the column Turn off the vacuum Ur Pwn gt Add no more than 700 uL DNA agarose solution from Step 5 to the MicroElute DNA Mini Column Turn on the vacuum source to draw the sample through the column Turn off the vacuum Repeat Steps 8 10 until all of the sample has been transferred to the column Add 300 ul Binding Buffer XP2 Turn on the vacuum source to draw the sample through the column Turn off
17. wing wash before proceeding to elution steps 23 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 Binding Buffer XP2 200 mL PDRO40 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 24
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