Gel Extraction
Contents
1. 60 C Note The yellow color of the Binding Buffer XP2 signifies a pH of lt 7 5 10 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 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 10 11 12 13 14 15 16 17 E Z N A Gel Extraction Kit Vacuum Protocol Incubate at 60 C for 7 minutes or until the gel has completely melted Vortex or shake the tube every 2 3 minutes Prepare the vacuum manifold according to manufacturer s instructions Connect the HiBind DNA Mini Column to the vacuum manifold Add no more than 700 uL DNA agarose solution from Step 5 to the HiBind 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 A2. Store DNA at 20 C 12 E Z N A Gel Extraction Kit Enzymatic Reaction Protocol E Z N A Gel Extraction Kit Protocol Purification of DNA from Enzymatic Reactions The following protocol is designed for DNA recovery from enzymatic reactions such as PCR and probe labeling reactions Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 13 000 x g e Vortexer Nuclease free 1 5 mL microcentrifuge tubes 100 ethanol Before starting e Prepare SPW Wash Buffer according to the Preparing Reagents section on Page 5 Note The yellow color of the Binding Buffer XP2 signifies a pH of lt 7 5 1 Determine the volume of the enzymatic reaction 2 Transfer the sample into a clean 1 5 mL microcentrifuge tube 3 Add 1 volume Binding Buffer XP2 4 Vortex or invert the sample to mix thoroughly 5 Briefly centrifuge the tube to collect any drops from the inside of the lid 6 Insert a HiBind DNA Mini Column into a 2 mL Collection Tube 7 Transfer the sample to the HiBind DNA Mini Column 8 Centrifuge at 10 000 x g for 1 minute at room temperature 13 E Z N A Gel Extraction Kit Enzymatic Reaction Protocol 9 10 11 12 13 14 T5 16 17 18 19 14 Discard the filtrate and reuse the collection tube Add 700 uL SPW Wash Buffer Note SPW Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 5 for instr
3. downstream manipulations The E Z N A Gel Extraction Kit uses proprietary chemistry and HiBind technology to recover DNA fragments between 70 bp and 20 kb with yields exceeding 85 The DNA band of interest is excised from the gel dissolved in Binding Buffer and transferred to a HiBind DNA Mini Column Following three rapid wash steps DNA is eluted with the Elution Buffer and is ready for other applications DNA is suitable for ligations PCR sequencing restriction digestion or various labeling reactions In addition this kit can be also used to recover DNA directly from enzymatic reactions such as PCR and enzyme digestion reactions Benefits of the E Z N A Gel Extraction Kit e Fast DNA recovery from agarose gel lt 10 minutes e Reliability Optimized buffers that guarantee pure DNA e Safety No organic extractions e Quality Purified DNA is suitable for most applications Q spin columns vs V spin columns The E Z N A Gel Extraction Kit is available with two different types of columns V spin columns have an attached cap while Q spin columns are capless The columns are otherwise identical in use and application Either column can be used with either the vacuum or centrifugation protocols D2500 is the V spin version of the Gel Extraction Kit while D2501 is the Q spin version Binding Capacity Each HiBind DNA Mini Column can bind 25 ug DNA Spin Protocol Vacuum Spin Protocol Excise DNA Band Excise DNA Ban
4. 7 minutes or until the gel has completely melted Vortex or shake the tube every 2 3 minutes E Z N A Gel Extraction Kit Spin Protocol Important Monitor the pH of the Gel Binding Buffer mixture after the gel has completely dissolved DNA yields will significantly decrease when the pH gt 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 6 Insert a HiBind DNA Mini Column in a 2 mL Collection Tube 7 Add no more than 700 uL DNA agarose solution from Step 5 to the HiBind DNA Mini Column 8 Centrifuge at 10 000 x g for 1 minute at room temperature 9 Discard the filtrate and reuse collection tube 10 Repeat Steps 7 9 until all of the sample has been transferred to the column 11 Add 300 uL Binding Buffer XP2 12 Centrifuge at maximum speed 213 000 x g for 1 minute at room temperature 13 Discard the filtrate and reuse collection tube 14 Add 700 uL SPW Wash Buffer Note SPW Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 5 for instructions 15 Centrifuge at maximum speed for 1 minute at room temperature 16 Discard the filtrate and reuse collection tube E Z N A Gel Extraction Kit Spin Protocol Optional Repeat Steps 14 16 for a second SPW Wash Buffer wash step Perform the second wash step for any salt sensitive downstre
5. E Z N A Gel Extraction Kit Table of Contents INTFOCUCTION Le eeescsccecssscssecccscscsecccccescsecccccescscsceesesesescecececesseees lllustrat d PO OO sesssresiuiiusessenucsuninseonu Kit Contents Storage and Stability scsecssseseecneeenes Preparing REAGENTS gaiiicmmiaccnacimunctamnmmsnwaanean Guideline for Vacuum Manifold cessecsccssecssecseeceeessees SPIN POCO CO hia ssesasotsresnerssseserecganteserarsearpaieasieiautennaeis MU ACUMUIITY PCE OCC a cose cee sccecexsxcsesecesesotssatesdentrceceneetstiececseenreens tenes Enzymatic Reaction ProtoCol sesessessessessessessessrerssssersesse Troubleshooting Guide essesseeseeseseresresrssresrrsrserssssssssess Order O ienai innna au R Re Manual Revision August 2012 N OMEGA bio tek Innovations in nucleic acid isolation Introduction 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 that 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 Gel purification of DNA is a common technique for the isolation of specific fragments from reaction mixtures However most methods either fail to completely remove agarose or shear DNA which can lead to problems in
6. am applications 17 Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications 18 Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube 19 Add 30 50 uL Elution Buffer or deionized water directly to the center of the column membrane Note The efficiency of eluting DNA from the HiBind DNA Mini Column is dependent on pH If eluting DNA with 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 70 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration 22 Store DNA at 20 C E Z N A Gel Extraction Kit Vacuum Protocol E Z N A Gel Extraction Kit Vacuum Protocol Materials and Equipment to be Supplied by User Vacuum Manifold See Page 6 Heat block or water bath capable of 60 C Microcentrifuge capable of at least 13 000 x g Vortexer Nuclease free 1 5 mL microcentrifuge tubes 100 ethanol Optional 5M Sodium Acetate pH 5 2 Optional Sterile deionized water Before starting Prepare the Vacuum Manifold See Page 6 Prepare SPW Wash Buffer according to the Preparing Reagents section on Page 5 Set heating block or water bath to
7. d from Gel from Gel Transfer Lysate amp Bind Transfer Lysate amp Bind Q Incubate and Melt Gel Incubate and Melt Gel with Binding Buffer with Binding Buffer Wash 3x 7 il 7 fl Wash 3x Dry Dry Elute Elute Kit Contents and Storage pot D2501 00 D2501 01 D2501 02 pwn gt s o w ren oa a Storage and Stability All E Z N A Gel Extraction Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature Please ensure that the bottle of Binding Buffer XP2 is tightly capped when not in use If any precipitates form in the buffers warm at 37 C to dissolve Preparing Reagents Dilute SPW Wash Buffer with 100 ethanol as follows and store at room temperature D2500 00 D2500 01 D2501 01 100 mL D2500 02 D2501 02 100 mL per bottle Guideline 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 Tors Tor Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Illustrated Vacuum Setup Omega Bio tek s VAC 08 C Vacuum Tubing D Vacu
8. dd 300 uL Binding Buffer XP2 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 100 ethanol prior to use Please see Page 5 for instructions Turn on the vacuum source to draw the sample through the column Turn off the vacuum 11 E Z N A Gel Extraction Kit Vacuum Protocol Optional Repeat Steps 15 17 for a second SPW Wash Buffer wash step Perform the second wash step for any salt sensitive downstream applications 18 Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube 19 Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications 20 Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube 21 Add 30 50 uL Elution Buffer or deionized water directly to the center of the column membrane Note The efficiency of eluting DNA from the HiBind DNA Mini Column is dependent on pH If eluting DNA with deionized water make sure that the pH is around 8 5 22 Let sit at room temperature for 2 minutes 23 Centrifuge at maximum speed for 1 minute Note This represents approximately 70 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration 24
9. gel to completely melt Add more Binding Buffer XP2 if necessary Inappropriate elution Use Elution Buffer or check the pH of the water used to buffer elute DNA With overuse TAE buffer loses its buffering capacity and its pH increases This raises the pH of the agarose DNA Binding Buffer solution which interferes with DNA binding to the HiBind matrix Adjust pH by adding 5 uL 5M sodium acetate pH 5 2 to the gel slice Use freshly prepared TAE buffer for gel purification in order to prevent the contamination of isolated DNA and improve yields Column clogged Agarose gel not Make sure water bath is set to 55 60 C and allow gel completely dissolved in to completely melt For large agarose slices gt 0 3 mL Binding Buffer XP2 it is recommended that the gel be diced into smaller fragments to aid melting No DNA eluted SPW Wash Buffer not Prepare SPW Wash Buffer as instructed on page 5 or as diluted with 100 ethanol indicated on bottle Incorrect amount of Measure the gel accurately and use 0 1 mL Binding Buffer Binding Buffer added XP2 per 0 1 g gel Agarose gel does not completely dissolve TAE TBE running buffer is not fresh Optical densities do not agree with DNA yield on agarose gel Make sure to wash column as instructed in Step 14 of the spin protocol and Step 15 of the vacuum protocol Alternatively rely on agarose gel ethidium bromide electrophoresis for quantization DNA sample floats out of well
10. uctions Centrifuge at maximum speed 213 000 x g for 1 minute at room temperature Discard the filtrate and reuse collection tube Repeat Steps 10 12 for a second SPW Wash Buffer wash step Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube Add 30 50 uL Elution Buffer or deionized water directly to the center of the column membrane Note The efficiency of eluting DNA from the HiBind DNA Mini Column is dependent on pH If eluting DNA with 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 70 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 800 832 8896 Possible Problems and Suggestions Low DNA Yields Too little Binding Buffer Volume of agarose gel slice determined incorrectly Add XP2 added to gel enough Binding Buffer XP2 as instructed Make sure water bath is set to 60 C and allow
11. um Source A Vacuum Manifold B Vacuum Flask E Z N A Gel Extraction Kit Spin Protocol E Z N A Gel Extraction Kit Spin Protocol Materials and Equipment to be Supplied by User Heat block or water bath capable of 60 C Microcentrifuge capable of at least 13 000 x g Vortexer e Nuclease free 1 5 mL microcentrifuge tubes 100 ethanol e Optional 5M Sodium Acetate pH 5 2 e Optional Sterile deionized water Before starting Prepare SPW Wash Buffer according to the Preparing Reagents section on Page 5 Set heating block or water bath to 60 C Note The yellow color of the Binding Buffer XP2 signifies a pH of lt 7 5 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 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
12. while loading agarose gel Ethanol not completely Centrifuge as instructed in Step 17 of the spin protocol removed from column and Step 19 of the vacuum protocol 15 Trace contaminants eluted from column increase Aso 16 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 Binding Buffer 200 mL PDR040 Binding Buffer 500 mL PDR041 SPW Wash Buffer 25 mL PDR045 2 mL Collection Tubes SS1 1370 00 1 5 mL DNase RNase free Centrifuge Tubes SS1 1210 0 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
Download Pdf Manuals
Related Search