E.Z.N.A.®Tissue DNA Kit - Omega Bio-Tek
Contents
1. and wash the cells once with cold PBS 4 C Resuspend cells in 200 uL PBS Proceed to Step 2 C For cells grown in a monolayer harvest the cell by either using a trypsin treatment or by scraping with a rubber policeman Wash cells twice with cold PBS 4 C Resuspend the cells in 200 uL PBS Proceed to Step 2 Add 25 uL OB Protease Solution Vortex to mix thoroughly Cultured Cells Protocol Optional Cultured cells have high levels of RNA which will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point 1 Add 4 uL RNase A 100 mg mL per 30 mg tissue 2 Let sit at room temperature for 2 minutes 3 Proceed to Step 3 below 3 Add 220 ul BL Buffer Note A wispy precipitate may form upon the addition of BL Buffer This is does not interfere with DNA recovery 4 Incubate at 70 C for 10 minutes Briefly vortex the tube once during incubation 5 Add 220 pL 100 ethanol Adjust the volume of ethanol required based on the amount of starting material Vortex to mix thoroughly 6 Inserta HiBind DNA Mini Column into a 2 mL Collection Tube 7 Transfer the entire sample from Step 5 to the HiBind DNA Mini Column including any precipitates that may have formed 8 Centrifuge at maximum speed 210 000 x g for 1 minute 9 Discard the filtrate and reuse the collection tube 10 Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Ple2. at room temperature for 12 months Proteinase Storage Buffer is no longer included in the kit Yield and Quality of DNA Determine the absorbance of an appropriate dilution 20 to 50 fold of the sample at 260 nm and then at 280 nm The DNA concentration is calculated as follows DNA concentration Absorbance 260 x 50 x Dilution Factor pg mL A value greater than 1 8 indicates greater than 90 nucleic acid Alternatively quantity as well as quality can sometimes best be determined by agarose gel ethidium bromide electrophoresis by comparison to DNA samples of known concentrations If necessary the DNA can be concentrated Add sodium chloride to reach a final concentration of 0 1M followed by 2X volumes 100 ethanol Mix well and incubate at 20 C for 10 minutes Centrifuge at 10 000 x g for 15 minutes and aspirate and discard the supernatant Add 700 uL 70 ethanol and centrifuge at 10 000 x g for 2 minutes Aspirate and discard the supernatant air dry the pellet for 2 minutes and resuspend the DNA in 20 uL sterile deionized water or 10 mM Tris HCl pH 8 5 Expected Yields HeLa Cells 1x 10 cells 5 6 ug Illustrated Protocol am O E O O AO a Lyse Adjust Binding Conditions Bind Wash 3X Dry Elute Kit Contents D3396 00 D3396 01 D3396 02 HiBind DNA Mini Columns Storage and Stability All E Z N A Tissue DNA Kit components are guaranteed for at least 12 months from the date of purchase whe
3. carried out using 50 100 uL Elution Buffer which slightly reduces overall DNA yield Volumes lower than 50 uL greatly reduce yields In some instances yields may be increased by incubating the column at 70 C rather than at room temperature upon the addition of Elution Buffer Store eluted DNA at 20 C 29 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 lysis time with TL Buffer and OB Incomplete lysis a Protease Solution If using more than 30 mg tissue increase Sample size is too large volumes of OB Protease TL Buffer BL Buffer and ethanol Sainoleisviscious Divide sample into multiple tubes and P adjust the volume to 250 uL with TL Buffer Incomplete F Completely homogenize sample Repeat elution with increased elution Poor elution volume Incubate columns at 70 C for 5 minutes with Elution Buffer Clogged Column DNA Wash Buffer must be diluted with 100 ethanol before use HBC Buffer must be diluted with isopropanol before use Low DNA iaa Overgrown culture contains lysed cells and Yield 9 degraded DNA Improper washing Increase starting material and volume of all Sample has low DNA reagents OB Protease TL Buffer BL Buffer content ethanol proportionally Load aliquots of lysate through the column successively Add 100 uL 3M NaOH to the column pr
4. ethanol Adjust the volume of ethanol required based on the amount of starting material Vortex to mix thoroughly Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Transfer the entire sample from Step 18 to the HiBind DNA Mini Column including any precipitates that may have formed Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the collection tube Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 6 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and collection tube Insert the HiBind DNA Mini Column into a new 2 mL Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with ethanol before use Please see Page 6 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the collection tube 30 31 32 33 34 35 36 37 Paraffin embedded Tissue Protocol Repeat Steps 27 29 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind 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 HiBind DNA Mini Column into a nuclease free 1 5 mL microcentrifuge tube Add 50 100 uL Elution Buffer heated to 70 C Let sit at room temperature for 2 minutes Centrifuge at maxim
5. sample every 20 30 minutes Lysis time depends on the amount and type of tissue used The average time is usually less than 3 hours Lysis can proceed overnight Optional Certain tissues such as liver tissue have high levels of RNA which will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point 10 11 1 Add 4 uL RNase A 100 mg mL per 30 mg tissue 2 Let sit at room temperature for 2 minutes 3 Proceed to Step 5 below Centrifuge at maximum speed 210 000 x g for 5 minutes Transfer the supernatant to a sterile 1 5 mL microcentrifuge tube Do not disturb or transfer any of the insoluble pellet Add 220 uL BL Buffer Adjust the volume of BL Buffer based on the amount of starting material Vortex to mix thoroughly Example If you used 400 uL of TL Buffer then add 420 uL BL Buffer and 420 uL 100 ethanol Note A wispy precipitate may form upon the addition of BL Buffer This is does not interfere with DNA recovery Incubate at 70 C for 10 minutes Add 220 uL 100 ethanol Adjust the volume of ethanol required based on the amount of starting material Vortex to mix thoroughly Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Transfer the entire sample from Step 9 to the HiBind DNA Mini Column including any precipitates that may have formed 12 13 14 13 16 IZ 18 19 20 21 22 10 Tissue DNA Proto
6. 02 OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z N A Tissue DNA Kit D3396 00 5 preps D3396 01 50 preps D3396 02 200 preps May 2013 For research use only Not intended for diagnostic testing E Z N A Tissue DNA Kit Table of Contents Introduction and OVEFVIEW cscsssscsscsecsssccsecseecssecseecseceneersees 2 Yield and Quality Of DNA isis ena 3 Mustiated ProtOCOlssuninmensnannsiamannmniienninaa 4 Kit Contents Storage and Stability sssecssesceecseeeneers 5 Preparing Reagan Sinarcas alcala 6 Recommended SettiNS cssecsesssessecsscseecseesesseessecsecseesseeseesses 7 Tissue Spin Protocol aaa das 8 Cultured Cells Spin Protocol cria 12 Mouse Tail Snips Spin Protoco l c ecmcmomommmmm 16 Paraffin embedded Tissue Spin Protoco l 20 Whole Blood and Body Fluids Protocol 24 Vacuum Spin PFOTOCOL ssscssssessovenssoscsnsssassovenssasrnoooneasesensnosnavoens 27 Troubleshooting GUICE rines 30 Ordena 31 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 certain optimal conditions allowing proteins and
7. Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 6 for instructions Switch on vacuum source to draw the HB Buffer 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 Page 6 for instructions Switch on vacuum source to draw the DNA Wash Buffer through the column Turn off the vacuum Repeat Steps 9 11 for a second DNA Wash step Remove the column from the vacuum manifold and transfer to a new 2 mL Collection Tube Centrifuge at maximum speed 210 000 x g for 2 minutes to completely dry the membrane Note It is important 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 16 17 18 19 20 Vacuum Protocol Add 50 200 uL Elution Buffer heated to 70 C Note Refer to individual protocols for recommended elution volumes Let sit at room temperature for 2 minutes Centrifuge at maximum speed for 1 minute Repeat Steps 16 18 for a second elution step Note Each 200 uL elution will typically yield of 60 70 of the DNA bound to the column Thus two elutions will generally yield 90 However increasing the elution volume will reduce the concentration of the final product To obtain DNA at higher concentrations elution can be
8. Buffer provided Add 25 uL OB Protease Solution Add 250 uL BL Buffer Vortex to mix thoroughly Note A wispy precipitate may form upon the addition of BL Buffer This is does not interfere with DNA recovery Blood and Body Fluids Protocol Optional RNA will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point 1 Add 4 uL RNase A 100 mg mL 2 Let sit at room temperature for 2 minutes 3 Proceed to Step 4 below 4 Incubate at 70 C for 10 minutes Briefly vortex the tube once during incubation 5 Add 250 uL 100 ethanol Vortex to mix thoroughly 6 Insert the HiBind DNA Mini Column into a 2 mL Collection Tube 7 Transfer the entire sample from Step 5 to the HiBind DNA Mini Column including any precipitates that may have formed 8 Centrifuge at maximum speed 210 000 x g for 1 minute 9 Discard the filtrate and reuse the collection tube 10 Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 6 for instructions 11 Centrifuge at maximum speed for 30 seconds 12 Discard the filtrate and collection tube 13 Insert the HiBind DNA Mini Column into a new 2 mL Collection Tube 25 14 15 16 17 18 19 20 21 22 23 Blood and Body Fluids Protocol Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with ethanol before use Please see Page 6 for in
9. ase see Page 6 for instructions 11 Centrifuge at maximum speed for 30 seconds 13 12 13 14 133 16 IZ 18 19 20 21 22 14 Cultured Cells Protocol Discard the filtrate and collection tube Insert the HiBind DNA Mini Column into a new 2 mL Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with ethanol before use Please see Page 6 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the collection tube Repeat Steps 14 16 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind 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 HiBind DNA Mini Column into a nuclease free 1 5 mL microcentrifuge tube Add 100 200 uL Elution Buffer heated to 70 C Let sit at room temperature for 2 minutes Centrifuge at maximum speed for 1 minute Cultured Cells Protocol 23 Repeat Steps 20 22 for a second elution step Note Each 200 uL elution will typically yield of 60 70 of the DNA bound to the column Thus two elutions will generally yield 90 However increasing the elution volume will reduce the concentration of the final product To obtain DNA at higher concentrations elution can be carried out using 50 100 uL Elution Buffer which slightly reduces overall DNA
10. card the filtrate and reuse the collection tube 12 Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 6 for instructions 17 13 14 15 16 17 18 19 20 21 22 23 18 Mouse Tail Snips Protocol Centrifuge at maximum speed for 30 seconds Discard the filtrate and collection tube Insert the HiBind DNA Mini Column into a new 2 mL Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with ethanol before use Please see Page 6 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the collection tube Repeat Steps 16 18 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind 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 HiBind DNA Mini Column into a nuclease free 1 5 mL microcentrifuge tube Add 100 200 uL Elution Buffer heated to 70 C Let sit at room temperature for 2 minutes Mouse Tail Snips Protocol 24 Centrifuge at maximum speed for 1 minute 25 Repeat Steps 22 24 for a second elution step Note Each 200 uL elution will typically yield of 60 70 of the DNA bound to the column Thus two elutions will generally yield 90 However increasing the elution volume will reduce the concentration of the fina
11. col Centrifuge at maximum speed for 1 minute Discard the filtrate and reuse the collection tube Add 500 uL HBC Buffer Note HBC Buffer must be diluted with isopropanol before use Please see Page 6 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and collection tube Insert the HiBind DNA Mini Column into a new 2 mL Collection Tube Add 700 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with ethanol before use Please see Page 6 for instructions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the collection tube Repeat Steps 18 20 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind 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 23 24 25 26 27 28 Tissue DNA Protocol Transfer the HiBind DNA Mini Column into a nuclease free 1 5 mL microcentrifuge tube Add 100 200 uL Elution Buffer heated to 70 C Let sit at room temperature for 2 minutes Centrifuge at maximum speed for 1 minute Repeat Steps 24 26 for a second elution step Note Each 200 uL elution will typically yield of 60 70 of the DNA bound to the column Thus two elutions will generally yield 90 However increasing the elution volume will reduce the concentration of the final product To obtain DNA at higher conc
12. ead of continuing with centrifugation follow the steps outlined below Note Please read through previous sections of this manual before beginning this protocol paying particular attention to the Materials and Equipment to be Supplied by User Materials and Equipment to be Supplied by User Vacuum Manifold recommend Cat VAC 08 Tabletop microcentrifuge capable of 13 000 x g Nuclease free 1 5 mL microcentrifuge tubes Shaking water bath heat block or incubator capable of 70 C Vortexer 100 ethanol Isopropanol Before Starting Set water bath heat block or incubator to 70 C Prepare DNA Wash Buffer and HBC Buffer according to the directions in the Preparing Reagents section on Page 6 Heat Elution Buffer to 70 C 1 Prepare samples by following one of the protocols above Tissue Protocol Page 8 Steps 1 9 Cultured Cells Protocol Page 12 Steps 1 5 Mouse Tail Snips Page 16 Steps 1 7 Paraffin embedded Tissue Page 20 Steps 1 18 Whole Blood and Body Fluids Page 24 Steps 1 5 Wk WN gt 2 Prepare the vacuum manifold according to manufacturer s instructions and connect the HiBind DNA Mini Column to the manifold 3 Transfer the entire sample to the HiBind DNA Mini Column including any precipitate that may have formed 27 10 11 12 13 14 15 28 Vacuum Protocol Switch on vacuum source to draw the sample through the column Turn off the vacuum
13. entrations elution can be carried out using 50 100 uL Elution Buffer which slightly reduces overall DNA yield Volumes lower than 50 uL greatly reduce yields In some instances yields may be increased by incubating the column at 70 C rather than at room temperature upon the addition of Elution Buffer Store eluted DNA at 20 C 11 Cultured Cells Protocol E Z N A Tissue DNA Kit Protocol Cultured Cells This protocol is designed for the rapid isolation of up to 25 ug genomic DNA from up to 5 x 10 cultured cells Materials and Equipment to be Supplied by User Tabletop microcentrifuge capable of 13 000 x g Nuclease free 1 5 mL microcentrifuge tubes Shaking water bath heat block or incubator capable of 70 C Vortexer 100 ethanol Isopropanol PBS Optional RNase stock solution 100 mg mL Before Starting 12 Set water bath heat block or incubator to 70 C Prepare DNA Wash Buffer and HBC Buffer according to the directions in the Preparing Reagents section on Page 6 Heat Elution Buffer to 70 C Chill PBS to 4 C Prepare the cell suspension using one of the following methods A Frozen cell samples should be thawed before starting this protocol Pellet the cells by centrifugation Wash the cells with cold PBS 4 C Resuspend cells in 200 uL PBS Proceed to Step 2 B For cells grown in suspension pellet 5 x 10 by spinning at 1 200 x gin a centrifuge tube Aspirate and discard the supernatant
14. ior Column matrix lost to loading the sample Centrifuge at 10 000 binding capacity during x g for 30 seconds Add 100 uL water to the storage columns and centrifuge at 10 000 x g for 30 seconds Discard the filtrate Resin from the column may be present in el uate Avoid centrifugation at speeds higher than specified The material can be remove from the eluate by centrifugation It will not interfere with PCR or restriction digests Extended centrifugation during elution Poor cell lysis due to incomplete mixing with BL Buffer Repeat the procedure make sure to vortex the sample thoroughly with BL Buffer Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 CTI ENT 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 PCRis a patented process of Hoffman La Roche Use of the PCR process requires a license 31 Notes 32
15. l product To obtain DNA at higher concentrations elution can be carried out using 50 100 uL Elution Buffer which slightly reduces overall DNA yield Volumes lower than 50 uL greatly reduce yields In some instances yields may be increased by incubating the column at 70 C rather than at room temperature upon the addition of Elution Buffer 26 Store eluted DNA at 20 C 19 Paraffin embedded Tissue Protocol E Z N A Tissue DNA Kit Protocol Paraffin embedded Tissue Materials and Equipment to be Supplied by User Tabletop microcentrifuge capable of 13 000 x g Nuclease free 1 5 mL microcentrifuge tubes Shaking water baths heat blocks or incubators capable of 37 90 C Vortexer Incubator 100 ethanol Isopropanol Xylene Optional RNase stock solution 100 mg mL Before Starting 20 Set water baths heat blocks or incubators to 37 C 55 C 70 C and 90 C Prepare DNA Wash Buffer and HBC Buffer according to the directions in the Preparing Reagents section on Page 6 Heat Elution Buffer to 70 C Place no more than 30 mg of tissue 2 mm in a nuclease free 2 mL microcentrifuge tube Add 1 mL xylene Vortex to mix thoroughly Centrifuge at maximum speed 210 000 x g for 10 minutes Aspirate and discard the supernatant without disturbing the pellet Add 1 mL 100 ethanol Centrifuge at maximum speed for 5 minutes Aspirate and discard the ethanol without disturbing the pellet Paraffin e
16. mbedded Tissue Protocol 8 Repeat Steps 5 7 for a second ethanol wash step 9 Dry the tissue pellet at 37 C for 15 minutes 10 Add 200 uL TL Buffer 11 Add 25 uL OB Protease Solution Vortex to mix thoroughly 12 Incubate at 55 C in a shaking water bath Note If a shaking water bath is not available vortex the sample every 20 30 minutes Lysis time depends on the amount and type of tissue used The average time is usually less than 3 hours Lysis can proceed overnight 13 Incubate at 90 C for 30 60 minutes Optional Certain tissues such as liver tissue have high levels of RNA which will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point 1 Add 4 uL RNase A 100 mg mL per 30 mg tissue 2 Let sit at room temperature for 2 minutes 3 Proceed to Step 14 below 14 Centrifuge at maximum speed for 5 minutes 15 Transfer the supernatant to a sterile 1 5 mL microcentrifuge tube Do not disturb or transfer any of the insoluble pellet 16 Add 220 uL BL Buffer Adjust the volume of BL Buffer based on the amount of starting material Vortex to mix thoroughly Note A wispy precipitate may form upon the addition of BL Buffer This is does not interfere with DNA recovery 17 Incubate at 70 C for 10 minutes 21 18 19 20 21 22 23 24 25 26 27 28 29 22 Paraffin embedded Tissue Protocol Add 220 uL 100
17. n stored as follows OB Protease Solution can be stored at room temperature for 12 months For long term storage gt 12 months store at 2 8 C Store all other components at room temperature 22 25 C Check buffers for precipitates before use Redissolve any precipitates by warming to 37 C Preparing Reagents Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D3396 02 100 mL per bottle Dilute HBC Buffer with isopropanol as follows and store at room temperature Check buffers for precipitation before use Redissolve any precipitates by warming to 37 C Recommended Settings 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 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 Conversion from millibars Multiply by Millimeters of mercury mm Hg 0 75 Inches of mercury inch Hg 0 0295 Tors Tort Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Illustrated Vacuum Setup Bge a Eo Omega Bio tek s VAC 08 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask Tissue DNA Protocol E Z N A Tissue DNA Kit Protocol Tis
18. other contaminants to be removed Nucleic acids are easily eluted with deionized water or a low salt buffer The E Z N A Tissue DNA Kit provides an easy and rapid method for the isolation of genomic DNA for consistent PCR and Southern analysis Up to 30 mg animal tissue mouse tail snips paraffin embedded tissue or 5 x 10 cultured cells can be readily processed This kit allows for the single or multiple simultaneous processing of samples There is no need for phenol chloroform extractions and time consuming steps are eliminated e g precipitation using isopropanol or ethanol Purified DNA can be directly used for most applications such as PCR Southern blotting and restriction enzyme digestion Benefits of the E Z N A Tissue DNA Kit Optimized buffers that guarantee pure DNA No organic extractions Purified DNA can be directly used for most downstream applications 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 The total number of 2 mL Collection Tubes has been reduced from 600 to 400 This change eliminates a transfer step and reduces waste OB Protease is now supplied in a liquid form eliminating the resuspension step to prior to use OB Protease Solution can also be stored
19. rtex the sample every 20 30 minutes Lysis time depends on the amount and type of tissue used Incubation time for complete tail lysis is dependent on tail length and animal age 0 5 cm tail pieces from a two week old mice will typically lyse in approximately 2 hours For older animals an overnight incubation may improve yields Bone and hair will not lyse Mouse Tail Snips Protocol Optional Mouse tail snips have low levels of RNA which will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point 1 Add 4 uL RNase A 100 mg mL per 30 mg tissue 2 Let sit at room temperature for 2 minutes 3 Proceed to Step 5 below 5 Centrifuge at maximum speed 10 000 x g for 5 minutes to pellet insoluble tissue debris and hair 6 Transfer the cleared lysate to a sterile 1 5 mL microcentrifuge tube Do not disturb or transfer any of the insoluble pellet 7 Add one volume BL Buffer and one volume 100 ethanol Vortex to mix thoroughly Example If you transfer 180 uL cleared lysate add 180 uL BL Buffer and 180 uL 100 ethanol Note A wispy precipitate may form upon the addition of BL Buffer This is does not interfere with DNA recovery 8 Inserta HiBind DNA Mini Column into a 2 mL Collection Tube 9 Transfer the entire sample from Step 7 to the HiBind DNA Mini Column including any precipitates that may have formed 10 Centrifuge at maximum speed for 1 minute 11 Dis
20. structions Centrifuge at maximum speed for 30 seconds Discard the filtrate and reuse the collection tube Repeat Steps 14 16 for a second DNA Wash Buffer wash step Centrifuge the empty HiBind 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 HiBind DNA Mini Column into a nuclease free 1 5 mL microcentrifuge tube Add 50 200 uL Elution Buffer heated to 70 C Let sit at room temperature for 2 minutes Centrifuge at maximum speed for 1 minute Repeat Steps 20 22 for a second elution step Note Each 200 uL elution will typically yield of 60 70 of the DNA bound to the column Thus two elutions will generally yield 90 However increasing the elution volume will reduce the concentration of the final product To obtain DNA at higher concentrations elution can be carried out using 50 100 uL Elution Buffer which slightly reduces overall DNA yield Volumes lower than 50 uL greatly reduce yields In some instances yields may be increased by incubating the column at 70 C rather than at room temperature upon the addition of Elution Buffer 24 Store eluted DNA at 20 C 26 Vacuum Protocol E Z N A Tissue DNA Kit Protocol Vacuum Spin Protocol Carry out disruption homogenization protease digestion and loading onto the HiBind DNA Mini Column as indicated in previous protocols Inst
21. sue This method is suitable for the isolation of DNA from up to 30 mg tissue Yields vary depending on source Optional Although no mechanical homogenization of tissue is necessary pulverizing the samples in liquid nitrogen will improve lysis and reduce incubation time Once the liquid nitrogen has evaporated transfer the powdered tissue into a clean 1 5 mL microcentrifuge tube Add 200 uL TL Buffer and proceed to Step 2 below Materials and Equipment to be Supplied by User Tabletop microcentrifuge capable of 13 000 x g Nuclease free 1 5 mL microcentrifuge tubes Shaking water baths heat blocks or incubators capable of 55 70 C Vortexer 100 ethanol e lsopropanol Optional RNase stock solution 100 mg mL Before Starting Set water baths heat blocks or incubators to 55 C and 70 C Prepare DNA Wash Buffer and HBC Buffer according to the directions in the Preparing Reagents section on Page 6 Heat Elution Buffer to 70 C 1 Mince up to 30 mg tissue and transfer in a 1 5 mL microcentrifuge tube 2 Add 200 uL TL Buffer Note In order to speed up lysis cut the tissue into small pieces For samples more than 30 mg simply scale up the volume of TL Buffer used for a 40 60 mg sample use 400 uL TL Buffer 3 Add 25 uL OB Protease Solution Vortex to mix thoroughly Tissue DNA Protocol Incubate at 55 C in a shaking water bath Note If a shaking water bath is not available vortex the
22. um speed for 1 minute Repeat Steps 33 35 for a second elution step Note Yields will depend on size and age of sample Certain samples may require prolonged lysis with TL Buffer Tissue fixed with paraformaldehyde will yield degraded DNA or RNA The extent of degradation depends on type of fixative used but the size of DNA obtained is usually less than 500 bp Degradation is not caused by the E Z N A Tissue DNA Protocol Store eluted DNA at 20 C 23 Blood and Body Fluids Protocol E Z N A Tissue DNA Kit Protocol Whole Blood and Body Fluids The procedure below has been optimized for the use with fresh or frozen blood samples of 11 250 uL in volume Anti coagulated blood saliva serum buffy coat or other body fluids also can be used Materials and Equipment to be Supplied by User Tabletop microcentrifuge capable of 13 000 x g Nuclease free 1 5 mL microcentrifuge tubes Shaking water baths heat blocks or incubators capable of 70 C Vortexer 100 ethanol Isopropanol Optional PBS Optional 10 mM Tris HCl Optional RNase stock solution 100 mg mL Before Starting 24 Set water bath heat block or incubator to 70 C Prepare DNA Wash Buffer and HBC Buffer according to the directions in the Preparing Reagents section on Page 6 Heat Elution Buffer to 70 C Transfer the sample into a nuclease free 1 5 mL microcentrifuge tube and bring the volume up to 250 uL with 10 mM Tris HCl PBS or Elution
23. yield Volumes lower than 50 uL greatly reduce yields In some instances yields may be increased by incubating the column at 70 C rather than at room temperature upon the addition of Elution Buffer 24 Store eluted DNA at 20 C 15 Mouse Tail Snips Protocol E Z N A Tissue DNA Kit Protocol Mouse Tail Snips Materials and Equipment to be Supplied by User Tabletop microcentrifuge capable of 13 000 x g Nuclease free 1 5 mL microcentrifuge tubes Shaking water baths heat blocks or incubators capable of 55 70 C Vortexer 100 ethanol Isopropanol Optional RNase stock solution 100 mg mL Before Starting 16 Set water baths heat blocks or incubators to 55 C and 70 C Prepare DNA Wash Buffer and HBC Buffer according to the directions in the Preparing Reagents section on Page 6 Heat Elution Buffer to 70 C Snip two pieces of mouse tail 0 2 0 5 cm in length and place into a nuclease free 1 5 mL microcentrifuge tube Note Follow all regulations regarding the safe and humane treatment of animals Mice should not be older than 6 weeks as lysis will be more difficult in older animals resulting in suboptimal DNA yields If possible obtain tail biopsies at 2 4 weeks and freeze samples at 70 C until DNA is extracted Add 200 uL TL Buffer Add 25 uL OB Protease Solution Vortex to mix thoroughly Incubate at 55 C for 1 4 hours in a shaking water bath Note If a shaking water bath is not available vo
Download Pdf Manuals
Related Search