E.Z.N.A.®Bacterial DNA Kit - Omega Bio-Tek
Contents
1. 10 11 12 13 14 Add 25 mg glass beads to 1 5 mL microcentrifuge tube Add sample to the glass beads Vortex at maximum speed for 5 minutes Let sample stand to allow the beads to settle Transfer supernatant to a new 1 5 mL microcentrifuge tube Nn gt gt Add 100 uL BTL Buffer and 20 uL Proteinase K Solution Vortex to mix thoroughly Incubate at 55 C in a shaking water bath Note Usually no more than 1 hour is required for bacterial lysis If a shaking water bath is not available incubate the samples and shake or briefly vortex every 20 30 minutes Add 5 uL RNase A Invert tube several times to mix Incubate at room temperature for 5 minutes Centrifuge at 10 000 x g for 2 minutes to pellet any undigested material Transfer the supernatant to a new 1 5 mL microcentrifuge tube Do not disturb the pellet Add 220 uL BDL Buffer Vortex to mix Incubate at 65 C for 10 minutes Note A wispy precipitate may form upon addition of BDL Buffer it does not interfere with DNA recovery 15 16 1 18 19 20 21 22 23 24 25 E Z N A Bacterial DNA Protocol Add 220 uL 100 ethanol Vortex for 20 seconds at maximum speed to mix thoroughly Note If any precipitate can be seen at this point break the precipitate by pipetting up and down 10 times Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Transfer the entire sample to the HiBind DNA2. Mini Column including any precipitate that may have formed Centrifuge at 10 000 x g for 1 minute Discard the filtrate and the collection tube Insert the HiBind DNA Mini Column into a new 2 mL Collection Tube Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 4 for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the collection tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol before use Please see Page 4 for instructions Centrifuge at 10 000 x g for 1 minute 26 27 28 29 30 31 32 33 34 E Z N A Bacterial DNA Protocol Discard the filtrate and reuse the collection tube Repeat Steps 24 26 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind DNA Mini Column at maximum speed gt 10 000 x g for 2 minutes to dry the column Note This step is critical for removal of trace ethanol that may interfere with downstream applications Insert the HiBind DNA Mini Column into a new nuclease free 1 5 mL microcentrifuge tube Add 50 100 uL Elution Buffer heated 65 C to the HiBind DNA Mini Column Note Make sure to add the Elution Buffer to the center of the HiBind matrix Each 50 100 uL elution typically yields 60 70 of the DNA bound to the HiBind matrix Two elutions generally yield 90 However increasing elution volume reduces the concen
3. be stored at room temperature for 12 months For long term storage gt 12 months store at 2 8 C Once reconstituted in buffer Lysozyme must be stored at 20 C Store RNase A at 2 8 C Store all other components at room temperature Check buffers for precipitates before use Redissolve any precipitates by warming to 37 C Preparing Reagents 1 Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D3350 02 100 mL to each bottle 2 Dissolve Lysozyme with Elution Buffer as follows and store at 20 C ee se mdded 3 Dilute HBC Buffer with isopropanol as follows and store at room temperature Guidelines for Vacuum Manifold The following is required for use with the Vacuum Protocol A Vacuum Manifold We recommend Omega Bio tek s VAC 08 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 08 200 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 vo N 94 Feen OMEGA Omega Bio tek s VAC 08 6 Vacuum Tubing A Vacuum Manifold D Vacuum Source B Vacuum Flask E Z N A
4. or 1 x 102 cell per spin column For larger volumes divide sample into multiple tubes Add more Lysozyme or extend the incubation time lt may be necessary to increase incubation by 15 minutes Incomplete removal of cell wall Repeat elution or increase elution volume see note on Page 9 Incubation of column at 65 C for 5 minutes after addition of Elution Buffer may increase yield Poor elution Saal MA DNA Wash Buffer must be diluted with 100 yield prop 9 ethanol Add 100 uL 3M NaOH to the column prior to loading the sample Centrifuge at 10 000 Column needs priming x g for 30 seconds Add 100 uL water to the columns and centrifuge at 10 000 x g for 30 seconds Discard the filtrate 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 14 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 HiBind DNA Mini Columns 200 columns DNACOL 02 BDL Buffer 100 mL 7064 15 Notes
5. Bacterial DNA Protocol E Z N A Bacterial DNA Protocol Centrifugation Protocol This method allows genomic bacterial isolation from up to 3 mL LB culture Materials and Equipment to be Supplied by User e Tabletop microcentrifuge e Nuclease free 1 5 mL microcentrifuge tubes Water bath capable of 37 C Shaking water bath capable of 55 C Incubator or water bath capable of 65 C 100 ethanol e Isopropanol TE Buffer Vortexer Before Starting Prepare DNA Wash Buffer HBC Buffer and lysozyme as instructed in the Preparing Reagents section on Page 4 Set an incubator or water bath to 65 C Seta water bath to 37 C Seta shaking water bath to 55 C Heat Elution Buffer to 65 C 1 Culture bacteria in LB media to log phase Overnight culture can be used in many cases 2 Centrifuge no more than 3 mL culture or 1 x 10 cells at 4 000 x g for 10 minutes at room temperature 3 Aspirate and discard the media 4 Add 100 uL TE Buffer Vortex to completely resuspend the pellet 5 Add 10 uL Lysozyme E Z N A Bacterial DNA Protocol Incubate at 37 C for 10 minutes Note The amount of enzyme required and or the length of incubation may need to be modified depending on the bacterial strain used Complete digestion of the cell wall is essential for efficient lysis Longer incubation time may yield better results Optional Follow the short protocol below for difficult to lyse bacteria
6. E Z N A Bacterial DNA Kit D3350 00 5 preps D3350 01 50 preps D3350 02 200 preps May 2013 E Z N A Bacterial DNA Kit Table of Contents Introduction and 0 01 00 lt 2 Kit Contents Storage and Stability 3 Preparing Resgents nes 4 Guidelines for Vacuum 0 3106610 4 444 44 4 4 4 4445 5 Bacterial DNA Centrifugation Protocol 6 Bacterial DNA Vacuum Protocol 10 Troubleshooting 6010 4 4 14 A 6 ea 15 Manual Revision May 2013 Fi OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction The E Z N A Bacterial DNA Kit allows rapid and reliable isolation of high quality total cellular DNA from a wide variety of gram positive and gram negative bacterial species Up to 1 x 10 bacterial cells can be processed The system combines the reversible nucleic acid binding properties of Omega Bio tek s HiBind matrix with the speed and versatility of spin column technology to yield up to 15 30 ug of DNA with an A A ratio of 1 7 1 9 Purified DNA is suitable for PCR restriction enzyme digestion and hybridization applications There are no organic extractions which reduces plastic waste and user time allowing multiple samples to be processed in parallel E Z N A Bacterial DNA Kit will isolate all cellular DNA including plasmid DNA Each HiBind DNA Mini Column can bind approximately 100 ug genomic DNA Using greater th
7. an 1 x 10 bacterial cells is not recommended Overview If using the E Z N A Bacterial DNA Kit for the first time please read this booklet to become familiar with the procedures Bacterial cells are grown to log phase and harvested The bacterial cell wall is removed by lysozyme digestion followed by Proteinase K digestion Following lysis binding conditions are adjusted and the sample is applied to a HiBind DNA spin column Three rapid wash steps remove trace salts and protein contaminants and DNA is eluted in water or low ionic strength buffer Purified DNA can be directly used in downstream applications without the need for further purification New in this Edition HB Buffer has been replaced by HBC Buffer Isopropanol is required and supplied by the user Equilibration Buffer used in the Troubleshooting section is no longer included with this kit Equilibration Buffer can be replaced with 3M NaOH provided by the user Kit Contents D3350 00 D3350 01 D3350 02 Purification 50 preps 200 preps HiBind DNA Mini Columns 5 50 0 1 0 20 40 BTL Buffer 20 mL BDL Buffer 20 mL HBC Buffer 25 mL DNA Wash Buffer 15 mL Glass Beads 2g Elution Buffer 15 mL Proteinase K Solution 1 5 mL 20 40 2 mL Collection Tubes 100 1 Storage and Stability All E Z N A Bacterial DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows Proteinase K Solution can
8. d the pellet Add 10 uL Lysozyme E Z N A Bacterial DNA Protocol Incubate at 37 C for 10 minutes Note The amount of enzyme required and or the length of incubation may need to be modified depending on the bacterial strain used Complete digestion of the cell wall is essential for efficient lysis Longer incubation time may yield better results Optional Follow the short protocol below for difficult to lyse bacteria 10 11 12 13 14 Add 25 mg glass beads to 1 5 mL microcentrifuge tube Add sample to the glass beads Vortex at maximum speed for 5 minutes Let sample stand to allow the beads to settle Transfer supernatant to a new 1 5 mL microcentrifuge tube Nn gt gt Add 100 uL BTL Buffer and 20 uL Proteinase K Solution Vortex to mix thoroughly Incubate at 55 C in a shaking water bath Note Usually no more than 1 hour is required for bacterial lysis If a shaking water bath is not available incubate the samples and shake or briefly vortex every 20 30 minutes Add 5 uL RNase A Invert tube several times to mix Incubate at room temperature for 5 minutes Centrifuge at 10 000 x g for 2 minutes to pellet any undigested material Transfer the supernatant to a new 1 5 mL microcentrifuge tube Do not disturb the pellet Add 220 uL BDL Buffer Vortex to mix Incubate at 65 C for 10 minutes Note A wispy precipitate may form upon addition of BDL Buffer it does not interfere wit
9. h DNA recovery 11 15 16 17 18 19 20 21 22 23 24 25 26 12 E Z N A Bacterial DNA Protocol Add 220 uL 100 ethanol Vortex for 20 seconds at maximum speed to mix thoroughly Note If any precipitate can be seen at this point break the precipitate by pipetting up and down 10 times Prepare the vacuum manifold according to manufacturer s instructions and connect the HiBind DNA Mini Column to the manifold Transfer the entire sample to the HiBind DNA Mini Column including any precipitate that may have formed Switch on vacuum source to draw the sample through the column Turn off the vacuum Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 4 for instructions Switch on vacuum source to draw the HBC Buffer through the column Turn off the vacuum Add 700 uL DNA Wash Buffer to the HiBind DNA Mini Column Note DNA Wash Buffer must be diluted with 100 ethanol before use Please see Page 4 for instructions Switch on vacuum source to draw the DNA Wash Buffer through the column Turn off the vacuum Repeat Steps 23 25 for a second DNA Wash step 27 28 29 30 31 32 33 34 Remove the column from the vacuum manifold and transfer to anew 2 ml collection tube provided with the kit Centrifuge at maximum speed 210 000 x g for 3 minutes to completely dry the membrane Note It is important
10. to dry the column membrane before elution Residual ethanol may interfere with downstream applications Insert the HiBind DNA Mini Column into a new nuclease free 1 5 mL microcentrifuge tube Add 50 100 uL Elution Buffer heated to 65 C to the HiBind DNA Mini Column Note Make sure to add the Elution Buffer to the center of the HiBind matrix Each 50 100 uL elution typically yields 60 70 of the DNA bound to the HiBind matrix Two elutions generally yield 90 However increasing elution volume reduces the concentration of the final product To obtain DNA at higher concentrations elution can be carried out using 50 uL Elution Buffer which slightly reduces overall DNA yield Volumes lower than 50 uL greatly reduce yields Let sit for 3 to 5 minutes at room temperature Note Yields may be increased by incubating the column at 65 C rather than at room temperature Centrifuge at 10 000 x g for 1 minute to elute the DNA Repeat Steps 30 32 for a second elution step Store eluted DNA at 20 C 13 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 Add the correct volume of BTL Buffer and incu Incomplete lysis bate at 55 C to obtain complete lysis lt may be necessary to extend incubation time to 1 hour Clogged Do not use greater than 3 mL culture at OD column Too much sample 10
11. tration of the final product To obtain DNA at higher concentrations elution can be carried out using 50 uL Elution Buffer which slightly reduces overall DNA yield Volumes lower than 50 uL greatly reduce yields Let sit for 3 to 5 minutes at room temperature Note Yields may be increased by incubating the column at 65 C rather than at room temperature Centrifuge at 10 000 x g for 1 minute to elute the DNA Repeat Steps 30 32 for a second elution step Store eluted DNA at 20 C E Z N A Bacterial DNA Protocol E Z N A Bacterial DNA Protocol Vacuum Protocol Materials and Equipment to be Supplied by User Tabletop microcentrifuge Nuclease free 1 5 mL microcentrifuge tubes Water bath capable of 37 C Shaking water bath capable of 55 C Incubator or water bath capable of 65 C 100 ethanol Isopropanol TE Buffer Vortexer Vacuum manifold with standard Luer adaptor Before Starting 10 Prepare DNA Wash Buffer HBC Buffer and lysozyme as instructed in the Preparing Reagents section on Page 4 Set an incubator or water bath to 65 C Set a water bath to 37 C Set a shaking water bath to 55 C Heat Elution Buffer to 65 C Culture bacteria in LB media to log phase Overnight culture can be used in many cases Centrifuge no more than 3 mL culture or 1 x 10 cells at 4 000 x g for 10 minutes at room temperature Aspirate and discard the media Add 100 uL TE Buffer Vortex to completely resuspen
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