E.Z.N.A.®Blood DNA Mini Kit - Omega Bio-Tek
Contents
1. Roche Use of the PCR process requires a license 19 Notes 202. into a 2 mL Collection Tube Transfer 750 uL sample to the column Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 9 11 until all the sample has been transferred to the column Place 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 5 for instructions Centrifuge at 210 000 x g for 1 minute Discard the filtrate and reuse Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol according to the instructions in the Preparing Reagents section on Page 5 Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 17 19 for a second DNA Wash Buffer wash step 21 22 23 24 25 26 27 E Z N A Blood DNA Mini Kit Protocols Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed 210 000 x g to dry the column matrix Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column into a nuclease free 2 mL microcentrifuge tube Add 100 200 uL Elution Buffer heated to 65 C Let sit at room temperature for 5 minutes Note Incubating the HiBind DNA Mini Column at 65 C rather than room temperature will give a modest increase i
3. the first elution this may increase yield while maintaining elution volume Store DNA at 20 C E Z N A Blood DNA Mini Kit Protocols E Z N A Blood DNA Mini Kit Protocol Buccal Swabs This protocol requires an increased volume of BL Buffer Fewer preparations can be performed Additional BL Buffer can be purchased separately Materials and Equipment to be Supplied by User e Tabletop microcentrifuge capable of at least 13 000 x g Nuclease free 2 mL microcentrifuge tubes Water bath incubator or heat block capable of 65 C 100 Ethanol e Isopropanol PBS Optional RNase stock solution when RNA free genomic DNA is required Before Starting Prepare DNA Wash Buffer and HBC Buffer according to the directions in the Preparing Reagents section on Page 5 Heat the Elution Buffer to 65 C 1 Place the Buccal Swab in a 2 mL microcentrifuge tube 2 Add 500 uL PBS Optional If RNA free genomic DNA is required add 5 uL RNase A 50 mg mL 3 Add 25 uL OB Protease Solution and 500 uL BL Buffer Vortex at maximum speed for 30 seconds 4 Incubate at 65 C for 10 minutes 5 Discard the buccal swab 6 Add 500 uL 100 ethanol Vortex at maximum speed for 20 seconds 7 Centrifuge briefly to collect any drops from the inside of the lid 10 11 12 13 14 15 16 17 18 19 20 10 E Z N A Blood DNA Mini Kit Protocols Insert a HiBind DNA Mini Column
4. A Mini Column for 2 minutes at maximum speed 210 000 x g to dry the column matrix Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column into a nuclease free 2 mL microcentrifuge tube Add 100 200 uL Elution Buffer heated to 65 C Let sit at room temperature for 5 minutes Note Incubating the HiBind DNA Mini Column at 65 C rather than room temperature will give a modest increase in DNA yield per elution Centrifuge at gt 13 000 x g for 1 minute Repeat Steps 24 26 for a second elution step Note Any combination of the following steps can be used to help increase DNA yield After adding the Elution Buffer incubate the column for 5 minutes e Increase the elution volume Repeat the elution step with fresh Elution Buffer this may increase the yield but decrease the concentration Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume Store DNA at 20 C Note Blood spots from finger pricks usually contain no more than 50 uL blood and yield approximately 500 ng to 1 ug DNA This is usually sufficient for PCR analysis To obtain higher DNA concentrations elute with 50 uL preheated elution buffer volumes lower than 50 uL greatly reduce yields Alternatively the first eluate can be used to perform a second elution E Z N A Blood
5. DNA Mini Kit Protocols E Z N A Blood DNA Mini Kit Protocol Buffy Coat The buffy coat fraction of whole blood is enriched with leukocytes and usually gives at least 5 fold more DNA than the same volume of blood To prepare the buffy coat from fresh whole blood simply centrifuge the sample at 3 000 4 000 x g for 10 minutes at room temperature Three layers should form a plasma upper layer a buffy coat middle layer and an erythrocyte bottom layer Carefully aspirate the plasma making sure not to disturb the layer of concentrated leukocytes The buffy coat can be drawn off with a pipette and used directly in the E Z N A Blood DNA Mini Kit or frozen at 70 C This protocol requires an increased volume of BL Buffer Fewer preparations can be performed Additional BL Buffer can be purchased separately Materials and Equipment to be Supplied by User Tabletop microcentrifuge capable of at least 13 000 x g Nuclease free 2 mL microcentrifuge tubes Water bath incubator or heat block capable of 65 C 100 Ethanol lsopropanol Optional 10mM Tris HCI or PBS Optional RNase stock solution when RNA free genomic DNA is required Before Starting Prepare DNA Wash Buffer and HBC Buffer according to the directions in the Preparing Reagents section on Page 5 Heat the Elution Buffer to 65 C 1 Transfer the sample into a sterile microcentrifuge tube and bring the volume up to 500 uL with 10mM Tris HCl PBS o
6. Discard the filtrate and reuse Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol according to the instructions in the Preparing Reagents section on Page 5 Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 14 16 for a second DNA Wash Buffer wash step 18 19 20 21 22 23 24 E Z N A Blood DNA Mini Kit Protocols Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed 210 000 x g to dry the column matrix Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column into a nuclease free 2 mL microcentrifuge tube Add 100 200 uL Elution Buffer heated to 65 C Let sit at room temperature for 5 minutes Note Incubating the HiBind DNA Mini Column at 65 C rather than room temperature will give a modest increase in DNA yield per elution Centrifuge at gt 13 000 x g for 1 minute Repeat Steps 20 22 for a second elution step Note Any combination of the following steps can be used to help increase DNA yield After adding the Elution Buffer incubate the column for 5 minutes Increase the elution volume Repeat the elution step with fresh Elution Buffer this may increase the yield but decrease the concentration Repeat the elution step using the eluate from
7. E Z N A Blood DNA Mini Kit D3392 00 5 preps D3392 01 50 preps D3392 02 200 preps May 2013 E Z N A Blood DNA Mini Kit Table of Contents Introduction and OVErVIEW cscssscsescnsesseccseccseessecssceseecseeeees Illustrated Protoeel nennen Kit Contents Storage and Stability Preparing REAQSIS nme Blood and Body Fluid Protocel nueuseenieneinen Buccal Swab Protocol isse Dried Blood Protocol cz renterne eden Buffy Coat PO nenne Troubleshooting Guide cu nen O aia Manual Revision May 2013 Fi 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 new HiBind matrix that specifically but reversibly binds DNA or RNA under optimal conditions allowing proteins and other contaminants to be removed Nucleic acids are easily eluted with deionized water or a low salt buffer The E Z N A Blood DNA Mini Kit provides an easy and rapid method for the isolation of genomic DNA for consistent PCR and Southern analysis Up to 250 uL of fresh frozen or anticoagulated whole blood can be readily processed at one time The E Z N A Blood DNA Mini Kit can also be used for the preparation of genomic DNA from buffy coat serum plasma saliva buccal swabs and other body fluids The E Z N A Blood DNA Kit allows
8. for single or multiple simultaneous processing of multiple samples There is no need for phenol chloroform extractions and time consuming steps are eliminated e g precipitation using isopropanol or ethanol Purified DNA obtained with the E Z N A Blood DNA Kit is ready for applications such as PCR restriction digestion and Southern blotting Benefits of the E Z N A Blood DNA Mini Kit Optimized buffers that guarantee pure DNA No organic extractions Purified DNA can be directly used in most downstream applications Binding Capacity Each HiBind DNA Mini Column can bind approximately 100 ug DNA Using greater than 250 uL whole blood or buffy coat is not recommended 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 e Equilibration Buffer can be replaced with 3M NaOH provided by the user OB Protease is now supplied in a liquid form eliminating the resuspension step to prior to use e OB Protease Solution can also be stored at room temperature for 12 months Proteinase Storage Buffer is no longer included in the kit Illustrated Protocol om O E O O LO E Lyse Adjust Binding Conditions Bind Wash 3X Dry Elute Kit Contents CTO NETO fantcorecientape m Storage and Stability All of the E Z N A Blood DNA Mini Ki
9. microcentrifuge tubes Water bath incubator or heat block capable of 65 C 100 Ethanol Isopropanol e Optional 10mM Tris HCI or PBS e Optional RNase stock solution when RNA free genomic DNA is required Before Starting Prepare DNA Wash Buffer and HBC Buffer according to the directions in the Preparing Reagents section on Page 5 Heat the Elution Buffer to 65 C 1 Transfer the sample into a sterile microcentrifuge tube and bring the volume up to 250 uL with 10mM Tris HCl PBS or Elution Buffer provided 2 Add 25 uL OB Protease Solution and 250 uL BL Buffer Vortex at maximum speed for 15 seconds Optional If RNA free genomic DNA is required add 5 uL RNase A 50 mg mL 3 Incubate at 65 C for 10 minutes Vortex briefly once during incubation 4 Add 260 uL 100 ethanol Vortex at maximum speed for 20 seconds 5 Centrifuge briefly to collect any drops from the inside of the lid 10 11 12 13 14 15 16 17 E Z N A Blood DNA Mini Kit Protocols Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Transfer the entire sample to the column Centrifuge at 210 000 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 5 for instructions Centrifuge at 210 000 x g for 1 minute
10. n DNA yield per elution Centrifuge at 213 000 x g for 1 minute Repeat Steps 23 25 for a second elution step Note Any combination of the following steps can be used to help increase DNA yield After adding the Elution Buffer incubate the column for 5 minutes Increase the elution volume Repeat the elution step with fresh Elution Buffer this may increase the yield but decrease the concentration Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume Store DNA at 20 C 11 E Z N A Blood DNA Mini Kit Protocols E Z N A Blood DNA Mini Kit Protocol Dried Blood Materials and Equipment to be Supplied by User Tabletop microcentrifuge capable of at least 13 000 x g Nuclease free 2 mL microcentrifuge tubes Water bath incubator or heat block capable of 65 C 100 Ethanol Isopropanol PBS Optional RNase stock solution when RNA free genomic DNA is required Before Starting 12 Prepare DNA Wash Buffer and HBC Buffer according to the directions in the Preparing Reagents section on Page 5 Heat the Elution Buffer to 65 C Cut or punch out the blood spot from the filter paper up to 200 uL blood can be used per spot Tear or cut the filter paper into small pieces and place them into a 2 mL nuclease free microcentrifuge tube Add 250 uL PBS Incubate at 65 C for 1 hour Vortex briefly every 20 minutes Add 25 uL OB Pro
11. od DNA Mini Kit Protocols Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed 210 000 x g to dry the column matrix Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column into a nuclease free 2 mL microcentrifuge tube Add 100 200 uL Elution Buffer heated to 65 C Let sit at room temperature for 5 minutes Note Incubating the HiBind DNA Mini Column at 65 C rather than room temperature will give a modest increase in DNA yield per elution Centrifuge at 213 000 x g for 1 minute Repeat Steps 21 23 for a second elution step Note Any combination of the following steps can be used to help increase DNA yield After adding the Elution Buffer incubate the column for 5 minutes Increase the elution volume Repeat the elution step with fresh Elution Buffer this may increase the yield but decrease the concentration Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume 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 Extend incubation time with BL Buffer and OB Incomplete Lysis Protease Solution Clogged Divide sample into multiple tubes and adj
12. r Elution Buffer provided 2 Add 25 uL OB Protease Solution and 500 uL BL Buffer Vortex at maximum speed for 15 seconds Optional If RNA free genomic DNA is required add 2 uL RNase A 50 mg mL 3 Incubate at 65 C for 10 minutes Vortex briefly once during incubation 4 Add 500 uL 100 ethanol Vortex at maximum speed for 20 seconds 15 10 11 12 13 14 13 16 17 18 16 E Z N A Blood DNA Mini Kit Protocols Centrifuge briefly to collect any drops from the inside of the lid Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Transfer the 750 uL sample to the column Centrifuge at 210 000 x g for 1 minute Discard the filtrate and the Collection Tube Repeat Steps 7 9 until all the sample has been transferred to the column 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 5 for instructions Centrifuge at 210 000 x g for 1 minute Discard the filtrate and reuse Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol according to the instructions in the Preparing Reagents section on Page 5 Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 15 17 for a second DNA Wash Buffer wash step 19 20 21 22 23 24 25 E Z N A Blo
13. t components are guaranteed for 12 months from the date of purchase when stored as follows OB Protease Solution can be stored at room temperature for 12 months For long term storage gt 12 months store OB Protease Solu tion at 2 8 C All other components should be stored at room temperature During ship ment or storage under cool ambient conditions a precipitate may form in the BL Buffer If a precipitate is present heat the bottle at 37 C to dissolve Preparing Reagents 1 Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D3392 02 100 mL per bottle 2 Dilute HBC Buffer with isopropanol as follows and store at room temperature E Z N A Blood DNA Mini Kit Protocols E Z N A Blood DNA Mini Kit Protocol Blood and Body Fluids The procedure below has been optimized for the use with fresh or frozen blood samples up to 250 uL in volume Anti coagulated blood saliva serum buffy coat or other body fluids can also be used In addition lt 10 of leukocytes or cultured cells may be used with this procedure For DNA extraction from tissue and mouse tail we suggest that you use the E Z N A Tissue DNA Kit Product No D3396 To isolate viral RNA from serum or other non cellular body fluids the E Z N A Viral RNA Kit Product No R6874 is recommended Materials and Equipment to be Supplied by User Tabletop microcentrifuge capable of at least 13 000 x g Nuclease free 2 mL
14. tease Solution Vortex at maximum speed for 15 seconds Incubate at 65 C for 30 minutes Vortex briefly several times during incubation Centrifuge at gt 13 000 x g for 5 minutes Transfer the supernatant to a nuclease free 2 mL microcentrifuge tube Add 1 volume BL Buffer and 1 volume 100 ethanol Vortex to mix thoroughly 10 11 12 13 14 15 16 17 18 19 20 21 E Z N A Blood DNA Mini Kit Protocols Centrifuge briefly to collect any drops from the inside of the lid Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Transfer the entire sample to the column Centrifuge at gt 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 5 for instructions Centrifuge at gt 10 000 x g for 1 minute Discard the filtrate and reuse Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol according to the instructions in the Preparing Reagents section on Page 5 Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 18 20 for a second DNA Wash Buffer wash step 13 22 23 24 25 26 27 28 14 E Z N A Blood DNA Mini Kit Protocols Centrifuge the empty HiBind DN
15. ust Column Too much sample the volume to 250 uL with BL Buffer eae ie Videos Divide sample into multiple tubes and adjust p the volume to 250 uL with BL Buffer Repeat elution with increased elution volume Poor Elution Incubate columns at 65 C for 5 minutes with Elution Buffer DNA Wash Buffer must be diluted with 100 ethanol before use If refrigerated DNA Wash Buffer must be brought to room temperature Improper Washing HBC Buffer must be diluted with isopropanol before use If refrigerated HBC Buffer must be Low DNA brought to room temperature Yield Increase starting material and volume of all Sample has low DNA reagents OB Protease BL Buffer ethanol Content proportionally Load aliquots of lysate through column successively Add 100 uL 3M NaOH to the column prior to loading the sample Let sit for 4 minutes Prime Columns Centrifuge at 10 000 x g for 60 seconds Add 100 uL water to the column and centrifuge at 10 000 x g for 60 seconds Discard the filtrate 18 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 2 mL DNase RNase free Microcentrifuge Tubes 1000 pk 10 pk cs SSI 1310 00 BL Buffer 100 mL PD062 DNA Wash Buffer 100 mL PS010 Elution Buffer 100 mL PDR048 RNase A 100 mg mL 2 5 mL RNA 03 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc PCR is a patented process of Hoffman La
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