E.Z.N.A.®BAC/PAC Maxi Kit - Omega Bio-Tek
Contents
1. media If you are using a frozen glycerol stock as the inoculum streak it onto an agar plate containing the appropriate antibiotic Inoculate a single colony into a 2 5 mL starter culture as described above 11 12 E Z N A BAC PAC DNA Maxi Kit Protocols Centrifuge 100 200 mL culture at 3 500 5 000 x g for 10 15 minutes at room temperature Decant or aspirate and discard the culture media Note 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 Add 16 mLT1 Buffer RNase A Vortex or pipet up and down to mix thoroughly Complete resuspension of cell pellet is vital for obtaining good yields Note RNase A must be added to T1 Buffer before use Please see the instructions in the Preparing Reagents section on Page 4 Add 16 mLT2 Buffer Invert and gently rotate the tube 10 20 times to obtain a clear lysate A 2 3 minute incubation may be necessary Note Avoid vigorous mixing as this will shear chromosomal DNA and lower BAC purity Do not allow the lysis reaction to proceed more than 5 minutes Store T2 Buffer tightly capped when not in use to avoid acidification from CO in the air Add 16 mL cold T3 Buffer Immediately invert several times until a flocculent white precipitate forms Note It is vital that the solution is mixed thoroughly and immediately after the addition of T3 Buffer to avoid localized precipitation If the mixture still appears vis2. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z N A BAC PAC Maxi Kit D2154 00 2 preps D2154 01 5 preps April 2013 For research use only Not intended for diagnostic testing E Z N A BAC PAC Maxi Kit Table of Contents Introduction and OVEFVIEW csccsccsscsecssccssecssecnsecseecseccneersees 2 Kit Contents Storage and Stability secsecssseceecseecseers 3 Preparing Reagents essseesssserssscerssseresseeessscesssseossseossnsssseseossss 4 Guidelines for Vacuum Manifold ssssssssssessssrsssssssssseseesssssss 5 BAC PAC DNA Maxi Kit Vacuum Protocol 6 BAC PAC DNA Maxi Kit Centrifugation Protocol 11 Alternative Protocol For BAC EluUtiOn ss ssseessssssssosseesress 16 Troubleshooting GUIGE i cscricscincerraninnceeannnnnnaanace 18 Manual Revision April 2013 N 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 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 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 E Z N A BAC PAC DNA Maxi Kit combines the power of HiBind technology with the time tested c
3. bed above E Z N A BAC PAC DNA Maxi Kit Protocols Centrifuge 100 200 mL culture at 3 500 5 000 x g for 10 15 minutes at room temperature Decant or aspirate and discard the culture media Note 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 Add 16 mLT1 Buffer RNase A Vortex or pipet up and down to mix thoroughly Com plete resuspension of cell pellet is vital for obtaining good yields Note RNase A must be added to T1 Buffer before use Please see the instructions in the Preparing Reagents section on Page 4 Add 16 mL T2 Buffer Invert and gently rotate the tube 10 20 times to obtain a clear lysate A 2 3 minute incubation may be necessary Note Avoid vigorous mixing as this will shear chromosomal DNA and lower BAC purity Do not allow the lysis reaction to proceed more than 5 minutes Store T2 Buffer tightly capped when not in use to avoid acidification from CO in the air Add 16 mL cold T3 Buffer Immediately invert several times until a flocculent white precipitate forms Note It is vital that the solution is mixed thoroughly and immediately after the addition of T3 Buffer to avoid localized precipitation If the mixture still appears viscous brownish or conglobated more mixing is required to completely neutralize the solution Incubate on ice for 10 minutes Centrifuge at 3 000 5 000 x g for 15 minutes at 4 C Note For faster re
4. cous brownish or conglobated more mixing is required to completely neutralize the solution Incubate on ice for 10 minutes Centrifuge at 3 000 5 000 x g for 15 minutes at 4 C Note For faster removal of the precipitated bacterial cell material one may order Omega s Lysate Clearance Filter Syringes to replace centrifugation step This filter cartridge completely removes SDS precipitates and clears bacterial lysates using a filter syringe 10 11 12 E Z N A BAC PAC DNA Maxi Kit Protocols Transfer the cleared supernatant by CAREFULLY aspirating it into an appropriate vessel Be careful not to disturb the pellet and that no cellular debris is transferred to the new tube Note When transferring the supernatant precipitates may float to the top of the supernatant Carefully insert the pipettor around the floating precipitates and transfer the cleared supernatant only Add 20 mL BAC Binding Buffer Invert the tube 20 times to mix thoroughly Note BAC Binding Buffer must be diluted with isopropanol prior to use Please see Page 4 for instructions Let sit at room temperature for 2 5 minutes Insert a HiBind BAC PAC DNA Maxi Column into a 50 mL Collection Tube provided Optional Protocol for Column Equilibration 13 14 15 16 Add 3 mL 3M NaOH to the HiBind BAC PAC DNA Maxi Column Let sit for 4 minutes at room temperature Centrifuge at 3 000 x g for 3 minutes Discard the filtrate and reuse t
5. e protocol below Optional To ensure the removal of residual ethanol from the column choose one of the following methods below to further dry the column before proceeding with DNA elution A Place the column into a vacuum container to dry for 15 minutes 1 Transfer the column into a vacuum chamber at room temperature Any device connected to a vacuum source may be used 2 Seal the chamber and apply vacuum for 15 minutes 3 Remove the column and proceed to Step 26 B Bake the column in a vacuum oven or incubator at 65 C for 10 minutes Remove the column and proceed to Step 26 26 Transfer the HiBind BAC PAC DNA Maxi Column to a 50 mL centrifuge tube not provided 27 Add 0 7 2 0 mL Elution Buffer sterile deionized water or TE buffer directly onto the column matrix 28 Let sit for 5 10 minutes at room temperature 29 Centrifuge at a maximum speed for 5 minutes 30 Store DNA at 20 C Note This represents approximately 70 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration The second elution may be performed using the first eluate to maintain a higher DNA concentration Heating the Elution Buffer to 65 C prior to the elution step may increase yields 10 E Z N A BAC PAC DNA Maxi Kit Protocols E Z N A BAC PAC Kit Centrifugation Protocol Materials and Equipment to be Supplied by User Refrigerated centrifuge with swing bucket rotor capable of at leas
6. er walls with viscous lysate Optical densities do not agree with DNA yield on agarose gel Make sure to wash column as instructed Alternatively rely on agarose gel ethidium bromide electrophoresis for quantitation RNA visible on agarose gel RNase A not added toT1 Buffer Over mixing of cell lysate upon addition of T2 Buffer Trace contaminants eluted from column increase Aso Add 1 vial of RNase to each bottle of T1 Buffer 18 Troubleshooting Guide BAC DNA floats out of well while loading agarose gel Ethanol not completely removed from column Centrifuge column as instructed to dry following wash steps BAC DNA will not perform in downstream application The column must be washed with ethanol and dried before elution Ethanol precipitation may be required following elution Traces of ethanol remain on column prior to elution As 60 Ago ratio is high or low SPM Wash Buffer was diluted with ethanol containing impurities Check the absorbency of the ethanol between 230 280 nm Do not use ethanol with high absorbency Purified BAC has RNA contamination Background reading is Centrifuge the purified DNA for 1 2 minutes transfer the high due to the silica fines sample to a new tube and measure the OD again Ensure that RNase A was added to T1 Buffer before use 19 Notes 20 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and Vacma
7. erature Preparing Reagents 1 Dilute BAC Binding Buffer with isopropanol as follows and store at room temperature 2 Dilute SPM Wash Buffer with 100 ethanol as follows and store at room temperature 3 Dilute HBC Buffer with isopropanol as follows and store at room temperature 4 Add the vial of RNase A to the bottle of T1 Buffer and store at 2 8 C 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 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 Tort Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Vacuum Setup ar a Haso Omega Bio tek s VAC 08 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask E Z N A BAC PAC DNA Maxi Kit Protocols E Z N A BAC PAC Kit Vacuum Protocol Materials and Equipment to be Supplied by User Refrigerated centrifuge with swing bucket rotor capable of at least 3 000 x g and capable of 4 C Vortexer Vacuum manifold Nuclease free 50 mL centrifuge tubes Falcon tubes recommended Ice bucket e lsopropanol 100 ethanol e Optional Incubato
8. fuge with swing bucket rotor capable of at least 3 000 x g and capable of 4 C Vortexer Nuclease free 50 mL centrifuge tubes Falcon tubes recommended Nuclease free 15 mL centrifuge tubes 70 ethanol 3M sodium acetate Ice bucket Optional Incubator capable of 65 C Optional Sterile deionized water or TE Buffer Before Starting gt Prepare ice bucket Chill 70 ethanol on ice Optional Set an incubator heat block or water bath to 65 C Optional Heat Elution Buffer to 65 C Transfer the HiBind BAC PAC DNA Maxi Column to a clean 50 mL centrifuge tube Add 3 mL Elution Buffer sterile deionized water or TE buffer directly onto the column matrix Let it sit at room temperature for 5 minutes Centrifuge at 4 000 x g for 5 minutes Note This represents approximately 70 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration The second elution may be performed using the first eluate to maintain a higher DNA concentration Heating the Elution Buffer to 65 C prior to the elution step may increase yields Dd 16 Carefully transfer the eluate to a nuclease free 15 mL centrifuge tube 10 11 12 13 14 E Z N A BAC PAC DNA Maxi Kit Protocols Add 1 10 volume 3M sodium acetate and 7 10 volume isopropanol Vortex to mix thoroughly Centrifuge at 5 000 x g for 20 minutes at 4 C Carefully decant and discard the supernatant without disturbing
9. he Collection Tube RWN gt Transfer 20 mL cleared supernatant from Step 11 by CAREFULLY aspirating it into the HiBind BAC PAC DNA Maxi Column Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind BAC PAC DNA Maxi Column Centrifuge at 4 000 x g for 5 minutes Discard the filtrate and reuse the Collection Tube Repeat Steps 13 15 until all of the cleared supernatant has been transferred to the HiBind BAC PAC DNA Maxi Column 13 17 18 19 20 21 22 23 14 E Z N A BAC PAC DNA Maxi Kit Protocols Add 10 mL HBC Buffer Note HBC Buffer must be diluted with isopropanol prior to use Please see Page 4 for instructions Centrifuge at 4 000 x g for 5 minutes Discard the filtrate and reuse the Collection Tube Add 20 mL SPM Wash Buffer Note SPM Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Centrifuge at 4 000 x g for 5 minutes Discard the filtrate and reuse the Collection Tube Centrifuge the empty HiBind BAC PAC DNA Maxi Column for 10 minutes at maximum speed to dry the column matrix Note It is important to dry the HiBind BAC PAC DNA Maxi Column matrix before elution Residual ethanol may interfere with downstream applications E Z N A BAC PAC DNA Maxi Kit Protocols Note For maximal yield and higher concentration of BAC DNA see the alterative elution protocol on Page 11 For the standa
10. l not to disturb the pellet and that no cellular debris is transferred to the HiBind BAC PAC DNA Maxi Column Turn on the vacuum source to draw the sample through the column Turn off the vacuum Repeat Steps 14 16 until all of the cleared supernatant has been transferred to the column 18 19 20 21 22 23 24 25 E Z N A BAC PAC DNA Maxi Kit Protocols Add 10 mL HBC Buffer Note HBC Buffer must be diluted with isopropanol prior to use Please see Page 4 for instructions Turn on the vacuum source to draw the HBC Buffer through the column Turn off the vacuum Add 20 mL SPM Wash Buffer Note SPM Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Turn on the vacuum source to draw the SPM Wash Buffer through the column Continue to apply the vacuum for 5 minutes after the SPM Wash Buffer has passed through the column Transfer the HiBind BAC PAC DNA Maxi Column to a 50 mL Collection Tube Centrifuge the empty HiBind BAC PAC DNA Maxi Column for 10 minutes at maximum speed to dry the column matrix Note It is important to dry the HiBind BAC PAC DNA Maxi Column matrix before elution Residual ethanol may interfere with downstream applications E Z N A BAC PAC DNA Maxi Kit Protocols Note For maximal yield and higher concentration of BAC DNA see the alterative elution protocol on Page 11 For the standard elution protocol proceed with th
11. moval of the precipitated bacterial cell material one may order Omega s Lysate Clearance Filter Syringes to replace centrifugation step This filter cartridge completely removes SDS precipitates and clears bacterial lysates using a filter syringe 10 11 12 13 E Z N A BAC PAC DNA Maxi Kit Protocols Transfer the cleared supernatant by CAREFULLY aspirating it into an appropriate vessel Be careful not to disturb the pellet and that no cellular debris is transferred to the new tube Note When transferring the supernatant precipitates may float to the top of the supernatant Carefully insert the pipettor around the floating precipitates and transfer the cleared supernatant only Add 20 mL BAC Binding Buffer Invert the tube 20 times to mix thoroughly Note BAC Binding Buffer must be diluted with isopropanol prior to use Please see Page 4 for instructions Let sit at room temperature for 2 5 minutes Prepare the vacuum manifold according to manufacturer s instructions Connect the HiBind BAC PAC DNA Maxi Column to the vacuum manifold Optional Protocol for Column Equilibration 14 15 16 T7 Add 3 mL 3M NaOH to the HiBind BAC PAC DNA Maxi Column Let sit for 4 minutes at room temperature Turn on the vacuum to draw the NaOH through the column Turn off the vacuum wn Transfer 20 mL cleared supernatant from Step 11 by CAREFULLY aspirating it into the HiBind BAC PAC DNA Maxi Column Be carefu
12. n 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. onsistency of alkaline SDS lysis of bacterial cells to deliver high quality BAC PAC DNA Omega Bio tek s HiBind BAC PAC DNA Maxi Columns facilitate the binding washing and elution steps thus enabling multiple samples to be simultaneously processed Yields vary according to copy number E coli strain and growth conditions Typically 200 mLovernight culture in an 2x YT medium typically produces 20 50 ug BAC DNA Isolated BAC PAC DNA is suitable for automated fluorescent DNA sequencing restriction enzyme digestion and other manipulations New in this Edition This manual has been edited for content and redesigned to enhance user readability e 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 HB Buffer has been replaced with HBC Buffer HBC Buffer must be diluted with isopropanol provided by user before use HB Buffer is no longer included with this kit Kit Contents Elution Buffer 10 mL 7 Elution Buffer is 10 mM Tris HCI pH 8 5 Storage and Stability All of the E Z N A BAC PAC Maxi DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows RNase A should be stored at 2 8 C T1 Buffer once RNase A is added should be stored at 2 8 C All remaining components should be stored at room temp
14. r capable of 65 C e Optional Sterile deionized water or TE Buffer e Optional 3M NaOH Before Starting Prepare T1 Buffer BAC Binding Buffer HBC Buffer and SPM Wash Buffer according to the Preparing Reagents section on Page 4 Prepare an ice bucket e Chill T3 Buffer on ice Optional Set an incubator heat block or water bath to 65 C e Optional Heat Elution Buffer to 65 C 1 Isolate a single colony from a freshly streaked selective plate and inoculate a culture of 100 200 mL 2xYT medium containing the appropriate antibiotic Incubate for 16 20 hours at 37 C with vigorous shaking 300 rpm Note Optimal growth conditions are vital in obtaining maximal BAC DNA yields To achieve the best conditions use a single colony from a freshly transformed or freshly streaked plate to inoculate a 2 5 mL starter culture containing the appropriate antibiotic Incubate for 8 hours at 37 C with vigorous shaking 300 rpm Use the starter culture to inoculate an appropriate volume of warm growth media containing the desired antibiotic Grow at 37 C for 16 20 hours with vigorous shaking 300 rpm Use a flask or vessel with a volume of at least 3 4 times the volume of the culture and dilute the starter culture 1 500 to 1 1000 into growth media If you are using a frozen glycerol stock as the inoculum streak it onto an agar plate containing the appropriate antibiotic Inoculate a single colony into a 2 5 mL starter culture as descri
15. rd elution protocol proceed with the protocol below Optional To ensure the removal of residual ethanol from the column choose one of the following methods below to further dry the column before proceeding with DNA elution A Place the column into a vacuum container to dry for 15 minutes 1 Transfer the column into a vacuum chamber at room temperature Any device connected to a vacuum source may be used 2 Seal the chamber and apply vacuum for 15 minutes 3 Remove the column and proceed to Step 26 B Bake the column in a vacuum oven or incubator at 65 C for 10 minutes Remove the column and proceed to Step 26 24 Transfer the HiBind BAC PAC DNA Maxi Column to a 50 mL centrifuge tube not provided 25 Add 0 7 2 0 mL Elution Buffer sterile deionized water or TE buffer directly onto the column matrix 26 Let sit for 5 10 minutes at room temperature 27 Centrifuge at a maximum speed for 5 minutes 28 Store DNA at 20 C Note This represents approximately 70 of bound DNA An optional second elution will yield any residual DNA though at a lower concentration The second elution may be performed using the first eluate to maintain a higher DNA concentration Heating the Elution Buffer to 65 C prior to the elution step may increase yields 15 E Z N A BAC PAC DNA Maxi Kit Protocols E Z N A BAC PAC Kit Alternative Protocol For BAC Elution Materials and Equipment to be Supplied by User Refrigerated centri
16. t 3 000 x g and capable of 4 C Vortexer e Vacuum manifold Nuclease free 50 mL centrifuge tubes Falcon tubes recommended e Ice bucket e lsopropanol 100 ethanol e Optional Incubator capable of 65 C e Optional Sterile deionized water or TE Buffer e Optional 3M NaOH Before Starting Prepare T1 Buffer BAC Binding Buffer HBC Buffer and SPM Wash Buffer according to the Preparing Reagents section on Page 4 Prepare an ice bucket Chill T3 Buffer on ice Optional Set an incubator heat block or water bath to 65 C e Optional Heat Elution Buffer to 65 C 1 Isolate a single colony from a freshly streaked selective plate and inoculate a culture of 100 200 mL 2xYT medium containing the appropriate antibiotic Incubate for 16 20 hours at 37 C with vigorous shaking 300 rpm Note Optimal growth conditions are vital in obtaining maximal BAC DNA yields To achieve the best conditions use a single colony from a freshly transformed or freshly streaked plate to inoculate a 2 5 mL starter culture containing the appropriate antibiotic Incubate for 8 hours at 37 C with vigorous shaking 300 rpm Use the starter culture to inoculate an appropriate volume of warm growth media containing the desired antibiotic Grow at 37 C for 16 20 hours with vigorous shaking 300 rpm Use a flask or vessel with a volume of at least 3 4 times the volume of the culture and dilute the starter culture 1 500 to 1 1000 into growth
17. the pellet Add 5 mL ice cold 70 ethanol Vortex to completely resuspend the pellet Centrifuge at 3 000 x g for 10 minutes Carefully decant and discard the supernatant without disturbing the pellet Let sit for 5 10 minutes to air dry Add 200 500 uL TE Buffer or sterile deionized water Store DNA at 20 C 17 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 Only use LB or YT medium containing ampicillin Do not use more than 200 mL with high copy BACs Cells may not be dispersed adequately prior to addi Poor cell lysis tion of T2 Buffer Vortex cell suspension to completely disperse Increase incubation time with T2 Buffer to obtain a clear lysate Do not incubate cultures for more than 16 hours at 37 C Storage of cultures for extended periods prior to BAC DNA isolation is detrimental No DNA eluted SPM Wash Buffer not Prepare SPM Wash Buffer according to the Preparing diluted with ethanol Reagents section on Page 4 Bacterial culture overgrown or not fresh HBC Buffer not diluted Prepare HBC Buffer according to the Preparing Reagents with isopropanol section on Page 4 High molecular weight DNA contamination of product Do not vortex or mix aggressively after adding T2 Buffer Adequate mixing is obtained by simply inverting and rotating tube to cov
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