Manual - Omega Bio-Tek
Contents
1. E Z 96 Tissue DNA Kit Protocols Seal the E Z 96 DNA Plate with AeraSeal film Centrifuge at 24 000 x g for 10 minutes Note Ensure that each sample has passed through the membrane in each well of the E Z 96 DNA Plate Longer centrifugation may be required if any lysate remains in any of the wells If some lysate is still left in the wells even with increased centrifugation time proceed to next step Remove and discard the AeraSeal film Add 500 uL HBC Buffer to each well Seal the plate with new AeraSeal film Note HBC Buffer must be diluted with isopropanol before use Please follow the instructions on Page 5 Centrifuge at 24 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate in the next step Remove and discard the AeraSeal film Add 600 uL DNA Wash Buffer to each well Seal the plate with new AeraSeal film Note DNA Wash Buffer must be diluted with 10096 ethanol before use Please follow the instructions on Page 5 Centrifuge at 24 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate in the next step Repeat Steps 12 14 for a second DNA Wash step Centrifuge at 24 000 x g for 15 minutes Discard the filtrate and the 96 well Square well Plate Note This step is critical for removing trace residual ethanol that might otherwise interfere with downstream applications The plate can be further dried by placing the plates in an incubator or vacuum oven pre2. 96 samples to be processed at one time Principle If using the E Z 96 Tissue DNA Kit for the first time please read this booklet to become familiar with the procedures Tissue or tail samples are cut into smaller pieces and then lysed in specially formulated buffer and protease Binding conditions are then adjusted and the sample is applied to the E Z 96 DNA Plate Three rapid wash steps remove trace contaminants such as residual polysaccharides and pure DNA is eluted with the Elution Buffer provided 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be directly used in downstream applications without the need for further purification New In this Edition HB Buffer has been replaced by HBC Buffer Isopropanol is required and supplied by the user Equilibration Buffer used in an Optional Column Equilibration protocol is no longer included with this kit Equilibration Buffer can be replaced with 3M NaOH provided by the user e OB Protease is now supplied in a liquid form eliminating the step to respuspend prior to use OB Protease Solution can also be stored at room temperature for 12 months Proteinase Storage Buffer is no longer included in the kit Centrifugation Protocol Vacuum Protocol Lyse the Samples Lyse the Samples Adjust the Binding Adjust the Binding Conditions Conditions Bind and Bind and Wash 3x Wash 3x Vacuum EE Dry Membrane Dry Membrane ERRA
3. Ensure that no visible pieces of tissue remain After applying to the plate wash with 300 uL of a 1 1 mixture of BL Buffer and ethanol and then with HBC Buffer and DNA Wash Buffer Mix thoroughly with BL Buffer prior to loading to the DNA plate Tissue sample must be cut or minced into small pieces Increase incubation time at 60 C with TL Buffer to ensure that tissue is completely lysed Before applying sample to column an aliquot of BL Buffer ethanol must be added See protocol above Dilute DNA Wash Buffer with the indicated volume of ethanol before use Dilute HBC Buffer with the indicated volume of isopropanol before use Repeat elution or increase elution volume see note on Page 11 Incubation at 70 C for 5 minutes with Elution Buffer may increase yields 27 28 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 BL Buffer 100 mL PD062 TL Buffer 100 mL PD061 DNA Wash Buffer 100 mL Elution Buffer 100 mL AeraSeal Film HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and Vacman are all trademarks of their respected companies PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license
4. Incubate at 60 C overnight or until the samples are completely lysed Mix occasionally during incubation by rotating the plate gently Note The lysate should be clear and viscous after digestion is complete Make sure the samples are completely lysed Undigested material may clog the E Z 96 DNA Plate in Step 11 5 Shake or vortex the plate vigorously from side to side do not shake up and down to avoid leaking around the caps Hold the caps to ensure the plate is sealed properly Ensure the lysate is completely homogeneous after shaking If a gelatinous mass is visible further digestion is required Optional Add 5 uL RNase A solution 20 mg mL not provided to each sample and let sit at room temperature for 5 minutes 6 Add two volumes BL Buffer about 450 uL to each sample A white precipitate may form at this step it will not interfere with DNA isolation Seal the plate with new Caps for Round well Plates Mix the sample by shaking or vortex the plate vigorously side to side for 1 minute Note BL Buffer must be diluted with 10096 ethanol before use Please follow the instructions on Page 5 7 Briefly spin the plate at 2 500 3 000 x g to collect any residue solution from the caps Note Do not centrifuge for a prolonged time Once the speed reaches 2 500 3 000 x g stop the centrifuge Optional Column Equilibration Protocol Place the E Z 96 DNA Plate on top of a 96 well Square well Plate provided Add 100 uL 3M NaOH into
5. Source review tables below for pressure settings Recommended Pressure mbar VAC 03 200 to 400 Conversion from millibars Multiply by Millimeters of Mercury mmHg 0 75 Kilopascals kPa 0 1 Inches of Mercury inchHg 0 0295 Tor or Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Illustrated Vacuum Setup EL Omega Bio tek s VAC 03 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask Guideline for Vacuum Manifold DNA Bind Setup Lysate Clearance Setup Lysate Clearance Plate E Z 96 DNA Plate X Vacuum Manifold Top Vacuum Manifold Top E Z 96 DNA Plate 300 uL Microplate Vacuum Manifold Bottom Vacuum Manifold Bottom Waste Collection c E Z 96 DNA Plate E Z 96 DNA Plate Vacuum Manifold Top Vacuum Manifold Top 96 well Racked Microtubes 300 uL Microplate 300 uL Microplate 96 well Racked Microtubes Vacuum Manifold Bottom Vacuum Manifold Bottom 7 E Z 96 Tissue DNA Kit Protocols E Z 96 Tissue DNA Kit Protocol Tissues and Mouse Tail 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 to each well of the 96 well Round well Plate supplied and proceed to Step 2 b
6. and reuse the 96 well Square well Plate Repeat Steps 15 17 for a second DNA Wash step Centrifuge at 24 000 x g for 15 minutes Discard the filtrate and the 96 well Square well Plate Note This step is critical for removing trace residual ethanol that might otherwise interfere with downstream applications The plate can be further dried by placing the plates in an incubator or vacuum oven preset at 70 C to dry the membrane 20 21 22 23 24 E Z 96 Tissue DNA Kit Protocols Transfer the E Z 96 DNA Plate to a set of 96 well Racked Microtubes provided Remove and discard the AeraSeal film Add 200 uL Elution Buffer heated at 70 C to each well of the E Z 96 DNA Plate Seal the E Z 96 DNA Plate with new AeraSeal film Let sit at room temperature for 2 5 minutes Centrifuge at gt 4 000 x g for 5 minutes Optional Repeat Steps 21 24 for a second elution step 25 Note A second elution will increase total DNA yield however due to increased elution volume the DNA concentration will be reduced If higher DNA concentration is desired the second elution can be performed with the 200 uL eluate from first elution reheated to 70 C Seal the 96 well Racked Microtubes with the Caps for Racked Microtubes and store the eluted DNA at 20 C 11 E Z 96 Tissue DNA Kit Protocols E Z 96 DNA Kit Protocol Cultured cells Materials and Equipment to be Supplied by User Centrifuge capable of 4 000 x g with
7. each well of the plate and let the plate sit for 4 minutes at room temperature Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the 96 well Square well Plate in the next step 8 Place the E Z 96 DNA Plate on top of a 96 well Square well Plate provided 9 Transfer all the lysate from Step 7 to each well of the E Z 96 DNA Plate 10 11 12 13 14 15 16 17 18 19 10 E Z 96 Tissue DNA Kit Protocols Seal the E Z 96 DNA Plate with AeraSeal film Centrifuge at gt 4 000 x g for 10 minutes Note Ensure that each sample has passed through the membrane in each well ofthe E Z 96 DNA Plate Longer centrifugation may be required if any lysate remains in any of the wells If some lysate is still left in the wells even with increased centrifugation time proceed to next step Remove and discard the AeraSeal film Add 500 uL HBC Buffer to each well Seal the plate with new AeraSeal film Note HBC Buffer must be diluted with isopropanol before use Please follow the instructions on Page 5 Centrifuge at gt 4 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Remove and discard the AeraSeal film Add 600 uL DNA Wash Buffer to each well Seal the plate with new AeraSeal film Note DNA Wash Buffer must be diluted with 10096 ethanol before use Please follow the instructions on Page 5 Centrifuge at 24 000 x g for 5 minutes Discard the filtrate
8. sample preparation Following lysis divide sample into Sample too viscous multiple tubes adjust volume to 250 uL with 10 mM Tris HCl DNA Wash Buffer must be diluted with Improper washing 10096 ethanol as instructed on Page 5 before use Incomplete Der due to BL Buffer is viscous and the sample must improper mixing with BL Washing leaves Buffer be vortexed thoroughly colored residue in column Ethanol was not added Dilute DNA Wash Buffer with the to the DNA Wash Buffer indicated volume of ethanol before use 26 Problem No DNA eluted Troubleshooting Guide IN ETT Extended centrifugation during elution step Poor cell lysis due to incomplete mixing with BL Buffer Incomplete cell lysis or protein degradation due to insufficient incubation Samples are rich in protein Poor cell lysis due to improper mixing with BL Buffer Poor cell and or protein lysis in Buffer TL Ethanol was not added to BL Buffer Ethanol was not added to the DNA Wash Buffer Isopropanol was not added to the HBC Buffer Poor elution Resin from the plate may be present in eluate Avoid centrifugation at speeds higher than specified The material can be removed from the eluate by centrifugation it will not interfere with PCR or restriction digestion Repeat the procedure and make sure to vortex the sample with BL Buffer immediately and completely Increase incubation time with TL Buffer and OB Protease Solution
9. E Z 96 Tissue DNA Kit D1196 00 1x 96 preps D1196 01 4x 96 preps D1196 02 20 x 96 preps May 2013 E Z 96 Tissue DNA Kit Table of Contents Introduction and PrInelplesuusei c i rore UR rta ERE Illustrated Protocol Sussie Kit Contents and Storage Preparing Reagan see Guideline for Vacuum Manifold sss Tissue and Mouse Tail Protocol sss Cultured Cell PROROEOL scatet itii didas 12 Bacterial DNA Protocols eene titii trt ian 16 Blood DNA Protocol ranas 20 Vacuum Manifold Protocol sse 24 Troubleshooting Sulde nenne 26 Ordering Information nn 28 Manual Revision May 2013 5 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Principle Introduction The E Z 96 Tissue DNA Kit allows rapid and reliable isolation of high quality total cellular DNA from a wide variety of animal tissues or cell cultures in a 96 well plate format Up to 30 mg tissue or two 0 6 cm mouse tail segments can be processed in each well The system combines the reversible nucleic acid binding properties of Omega Bio tek s HiBind matrix with the speed and versatility of E Z 96 DNA Plate to eliminate polysaccharides phenolic compounds and enzyme inhibitors from tissue lysates Purified DNA is suitable for PCR restriction digestion and hybridization applications There are no organic extractions thus reducing plastic waste and hands on time to allow up to
10. RE Elute Elute Kit Contents fezesonarare 9 6 welRacked mirorubes 129 Caps for Racked Microtubes 250x8 OB Protease Solution Storage and Stability All of the E Z 96 Tissue DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows OB Protease Solution can be stored at room temperature for up to 12 months For long term storage store OB Protease Solution at 2 8 C During shipment or storage in cool ambient conditions precipitates may form in the buffers Dissolve such deposits by warming the solution at 37 C and gently shaking Preparing Reagents Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D1196 00 100 mL D1196 01 400 mL D1196 02 800 mL per bottle Dilute HBC Buffer with isopropanol as follows and store at room temperature O areas added For Protocols other than Blood dilute BL Buffer with 10096 ethanol as follows and store at room temperature D1196 01 100 mL D1196 02 500mL Important DO NOT dilute BL Buffer with ethanol if the Blood Protocol is to be used Guideline for Vacuum Manifold The following is required for use with the Vacuum Protocol A Vacuum Manifold We recommend Omega Bio tek s VAC 03 Other Compatible Vacuum Manifolds Qiagen OlAvac24 Sigma AldrichVM20 Promega Vacman or manifold with standard Luer connector B Vacuum Flask C Vacuum Tubing D Vacuum
11. and reuse the 96 well Square well Plate in the next step Remove and discard the AeraSeal film Add 600 uL DNA Wash Buffer to each well Seal the plate with new AeraSeal film Note DNA Wash Buffer must be diluted with 100 ethanol before use Please follow the instructions on Page 5 Centrifuge at gt 4 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate in the next step Repeat Steps 14 16 for a second DNA Wash step Centrifuge at 24 000 x g for 15 minutes Discard the filtrate and the 96 well Square well Plate Note This step is critical for removing trace residual ethanol that might otherwise interfere with downstream applications The plate can be further dried by placing the plates in an incubator or vacuum oven preset at 70 C to dry the membrane 19 20 21 22 23 E Z 96 Tissue DNA Kit Protocols Transfer the E Z 96 DNA Plate to the 96 well Racked Microtubes provided Remove and discard the AeraSeal film Add 200 uL Elution Buffer heated at 70 C to each well of the E Z 96 DNA Plate Seal the E Z 96 DNA Plate with new AeraSeal film Let sit at room temperature for 2 5 minutes Centrifuge at gt 4 000 x g for 5 minutes Optional Repeat Steps 20 23 for a second elution step 24 Note A second elution will increase total DNA yield however due to increased elution volume the DNA concentration will be reduced If higher DNA concentration is desired the second
12. apply the vacuum for 10 minutes after all liquid has passed through the E Z 96 DNA Plate Place the plate in a vacuum oven preset on 70 C to further dry the plate It is very critical to completely dry the plate before elution Place the 96 well Racked Microtubes inside the base of the manifold Place the E Z 96 DNA Plate on top of the manifold Add 200 uL Elution Buffer heated to 70 C to each well Turn on the vacuum source to draw the liquid completely through the E Z 96 DNA Plate Seal the 96 well Racked Microtubes with the Caps for Racked Microtubes and store the eluted DNA at 20 C 25 Troubleshooting Guide Please use this guide to solve any problems that may arise We hope that it will aid in clearing up any questions for you If for any reason you need further assistance please contact our technical support staff at our Toll Free Number 1 800 832 8896 Possible Problems and Suggestions Extend lysis incubation time with TL Buffer and OB protease Solution Add the correct volume of BL Buffer and incubate for specified time at 60 C Incomplete lysis If using more than 30 mg tissue increase proportionately volumes of OB Protease Solution TL Buffer BL Buffer and ethanol Pass aliquots of lysate through successively Sample amount too large 96 well plate is clogged NEM Increase the centrifugation time by an additional 10 minutes Or add HBC Buffer and proceed with next step Incomplete lysis from
13. ater bath to 56 C Set a water baths incubators or heat blocks to 30 C Heat Elution Buffer to 70 C Collect and resuspend the bacteria using one of the following protocols A For2mL gram positive bacteria culture Centrifuge bacteria at 4 000 x g for 10 minutes Remove and discard the supernatant Add 180 uL TE Buffer and resuspend the pellet Add 18 uL lysozyme 50 mg mL and vortex to mix Incubate at 30 C for 10 minutes Centrifuge digested cell at 4 000 x g for 5 minutes Aspirate supernatant leaving 20 uL residual liquid Vortex to resuspend pellet Continue the protocol with Step 2 Oo ONAMRWN gt B For 2 mL gram negative bacteria culture 1 Centrifuge bacteria at 4 000 x g for 10 minutes 2 Remove and discard the supernatant 3 Add 20 uL TE Buffer and resuspend the pellet 4 Continue the protocol with Step 2 E Z 96 Tissue DNA Kit Protocols Add 200 uL TL Buffer and 25 uL OB Protease Solution Seal with mat or caps Vortex to mix thoroughly Note TL Buffer and OB Protease Solution can be made as a mastermix before adding Incubate at 56 C in a shaking water bath to complete lysis Note Usually no more than 1 hour is required for bacterial lysis If no shaking water bath is available incubate and shake or briefly vortex the samples every 20 30 minutes Add 5 uL RNase A 20 mg mL not provided Vortex to mix Let sit at room temperature for 5 minutes Add 450 uL BL Buffer A precipitate may form at
14. e interfere with downstream applications The plate can be further dried by placing the plates in an incubator or vacuum oven preset at 70 C to dry the membrane Transfer the E Z 96 DNA Plate to the 96 well Racked Microtubes provided Remove and discard the AeraSeal film E Z 96 Tissue DNA Kit Protocols 23 Add 200 uL Elution Buffer heated at 70 C to each well of the E Z 96 DNA Plate Seal the E Z 96 DNA Plate with new AeraSeal film 24 Let sit at room temperature for 2 5 minutes 25 Centrifuge at 24 000 x g for 5 minutes Optional Repeat Steps 22 25 for a second elution step Note A second elution will increase total DNA yield however due to increased elution volume the DNA concentration will be reduced If higher DNA concentration is desired the second elution can be performed with the 200 uL eluate from first elution reheated to 70 C 26 Sealthe 96 well Racked Microtubes with the Caps for Racked Microtubes and store the eluted DNA at 20 C 23 E Z 96 Tissue DNA Kit Protocols E Z 96 DNA Kit Protocol Vacuum Protocol The following protocol has been tested only on cultured cells and limited types of animal tissues It may not work for some types of animal tissue samples rich in polysaccharides Materials and Equipment to be Supplied by User Vacuum Manifold 1 Prepare the lysate using one of the previous protocols E Z 96 Tissue Protocol Steps 1 7 E Z 96 Cultured Cells Protocol Steps 1 4 Bacterial DNA Pro
15. elow Note Do not use too much starting material the lysate will be too viscous and may clog the E Z 96 DNA Plate Materials and Equipment to be Supplied by User e Centrifuge capable of 4 000 x g with swing bucket rotor Centrifuge adapter for deep well microplates Water baths incubators or heat blocks capable of 70 C 10096 ethanol Isopropanol Multichannel pipet with tips Vortexer Optional RNase A 20 mg mL e Optional Liquid nitrogen for freezing disrupting samples Optional 3M NaOH Before Starting Prepare reagents according to Preparing Reagents section on Page 5 Set a water bath incubator or heat block to 60 C Heat Elution Buffer to 70 C 1 Mince 20 mg tissue and place into the 96 well Round well Plate provided For mouse tails cut the samples to 0 6 cm pieces for rat tails cut the samples to 0 3 cm pieces Place two pieces into each well 2 Add 200 uL TL Buffer and 25 uL OB Protease Solution Seal with Caps for Round well Plates Vortex to mix thoroughly Note It is very important that samples are completely submerged in the solution If the TL Buffer does not completely cover the sample increase the sample volume to 300 uL Additional reagent can be purchased separately TL Buffer and OB Protease can be made as a mastermix before adding E Z 96 Tissue DNA Kit Protocols 3 Briefly spin the plate at 2 500 3 000 x g to collect any residue solution from the caps 4
16. elution can be performed with the 200 uL eluate from first elution reheated to 70 C Seal the 96 well Racked Microtubes with the Caps for Racked Microtubes and store the eluted DNA at 20 C 19 E Z 96 Tissue DNA Kit Protocols E Z 96 DNA Kit Protocol Blood Important DO NOT dilute BL Buffer with ethanol prior to performing this protocol Materials and Equipment to be Supplied by User Centrifuge capable of 4 000 x g with a swing bucket rotor Adapter for deep well microplates Water bath incubator or heat block capable of 60 C 10096 ethanol Isopropanol Multichannel pipet with tips Incubator or vacuum oven preset at 70 C Vortexer Optional RNase A 20 mg mL Optional 3M NaOH Before Starting 20 Prepare reagents according to Preparing Reagents section on Page 5 Set a water bath incubator or heat block to 60 C Heat Elution Buffer to 70 C Pipet 25 uL OB Protease Solution into the bottom of each well of a 96 well Round well Plate provided Add 250 uL whole blood serum or body fluid to each well of the 96 well Round well Plate Up to 6 x 10 lymphocytes can be used in each well Note For sample volumes smaller or larger than 250 pL adjust the sample volume to 250 uL Add 250 uL BL Buffer A white precipitate may form at this step it will not interfere with DNA isolation Seal the plate with Caps for Round well Plates Mix the samples by shaking or vortex the plate vigorously side to side fo
17. ng around the caps Hold the caps to ensure the plate is sealed properly Ensure the lysate is completely homogeneous after shaking If a gelatinous mass is visible further digestion is required Optional Add 5 uL RNase A solution 20 mg mL not provided to each sample and let sit at room temperature for 5 minutes 3 Add two volumes BL Buffer about 450 uL to each sample A white precipitate may form at this step it will not interfere with DNA isolation Seal the plate with new Caps for Round well Plates Mix the sample by shaking or vortex the plate vigorously side to side for 1 minute Note BL Buffer must be diluted with 10096 ethanol before use Please follow the instructions on Page 5 4 Briefly spin the plate at 2 500 3 000 x g to collect any residue solution from the caps Note Do not centrifuge for a prolonged time Once the speed reaches 2 500 3 000 x g stop the centrifuge Optional Column Equilibration Protocol Place the E Z 96 DNA Plate on top of a 96 well Square well Plate provided Add 100 uL 3M NaOH into each well of the plate and let the plate sit for 4 minutes at room temperature Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the 96 well Square well Plate in the next step 5 Place the E Z 96 DNA Plate on top of a 96 well Square well Plate provided 6 Transfer all the lysate from Step 4 to each well of the E Z 96 DNA Plate 13 10 11 12 13 14 T5 16 14
18. p 8 to each well of the E Z 96 DNA Plate 11 Seal the E Z 96 DNA Plate with AeraSeal film 12 Centrifuge at 24 000 x g for 10 minutes Note Ensure that each sample has passed through the membrane in each well of the E Z 96 DNA Plate Longer centrifugation may be required if any lysate remains in any of the wells If some lysate is still left in the wells even with increased centrifugation time proceed to next step 21 13 14 15 16 17 18 19 20 21 22 22 E Z 96 Tissue DNA Kit Protocols Remove and discard the AeraSeal film Add 500 uL HBC Buffer to each well Seal the plate with new AeraSeal film Note HBC Buffer must be diluted with isopropanol before use Please follow the instructions on Page 5 Centrifuge at gt 4 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate in the next step Remove and discard the AeraSeal film Add 600 uL DNA Wash Buffer to each well Seal the plate with new AeraSeal film Note DNA Wash Buffer must be diluted with 100 ethanol before use Please follow the instructions on Page 5 Centrifuge at 24 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate in the next step Repeat Steps 16 18 for a second DNA Wash step Centrifuge at 24 000 x g for 15 minutes Discard the filtrate and the 96 well Square well Plate Note This step is critical for removing trace residual ethanol that might otherwis
19. r 1 minute Note DO NOT dilute BL Buffer with ethanol prior to performing this protocol Shake the plate side to side not up and down to prevent possible leakage around caps E Z 96 Tissue DNA Kit Protocols Optional Add 20 uL RNase A solution 20 mg mL not provided to each sample and let sit at room temperature for 5 minutes 4 Centrifuge briefly at 2 500 3 000 x g to collect any solution from the caps 5 Incubate at 60 C for 10 minutes in an incubator or oven Mix occasionally during incubation by rotating the plate gently Note Incubation for more than 30 minutes at 60 C can cause DNA degradation 6 Centrifuge briefly at 2 500 3 000 x g to collect any solution from the caps Remove the caps and add 250 uL 10096 ethanol to each well 7 Sealthe 96 well Round well Plate provided using new Caps for Round well Plates 8 Mixthe samples by vortexing or vigorously shaking the plate side to side for 1 minute Centrifuge briefly at 3 000 rpm to collect any liquid from the caps Optional Column Equilibration Protocol Place the E Z 96 DNA Plate on top of a 96 well Square well Plate provided Add 100 uL 3M NaOH into each well of the plate and let the plate sit for 4 minutes at room temperature Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the 96 well Square well Plate in the next step 9 Place the E Z 96 DNA Plate on top of a 96 well Square well Plate provided 10 Transfer all the lysate from Ste
20. set at 70 C to dry the membrane 17 18 19 20 21 E Z 96 Tissue DNA Kit Protocols Transfer the E Z 96 DNA Plate to the 96 well Racked Microtubes provided Remove and discard the AeraSeal film Add 200 uL Elution Buffer heated at 70 C to each well of the E Z 96 DNA Plate Seal the E Z 96 DNA Plate with new AeraSeal film Let sit at room temperature for 2 5 minutes Centrifuge at gt 4 000 x g for 5 minutes Optional Repeat Steps 18 21 for a second elution step 22 Note A second elution will increase total DNA yield however due to increased elution volume the DNA concentration will be reduced If higher DNA concentration is desired the second elution can be performed with the 200 uL eluate from first elution reheated to 70 C Seal the 96 well Racked Microtubes with the Caps for Racked Microtubes and store the eluted DNA at 20 C 15 E Z 96 Tissue DNA Kit Protocols E Z 96 DNA Kit Protocol Bacteria Materials and Equipment to be Supplied by User Centrifuge capable of 4 000 x g equipped with swinging bucket rotor Adapter for deep well microplates 100 ethanol Isopropanol Water baths incubators or heat blocks capable of 70 C Shaking water bath capable of 56 C TE Buffer Lysozyme 50 mg mL RNase A 20 mg mL Vortexer Deep well culture plate with mat or caps Optional 3M NaOH Before Starting 16 Prepare reagents according to Preparing Reagent section Set a shaking w
21. swing bucket rotor Adapter for deep well microplates Water baths incubators or heat blocks capable of 70 C 100 ethanol Isopropanol Multichannel pipet with tips Ice bucket Vortexer Trypsin PBS Optional RNase A 20 mg mL Optional 3M NaOH Before Starting 12 Prepare reagents according to Preparing Reagents section on Page 5 Set a water baths incubators or heat blocks to 60 C Heat Elution Buffer to 70 C Chill PBS on ice Harvest and resuspend the cells using one of the following protocols A For cells grown in suspension do not use more than 5 x 10 cells 1 Centrifuge cells at 300 x g for 5 minutes 2 Resuspend cells with 200 uL cold 4 C PBS and transfer to a 96 well Round well Plate provided 3 Add 25 uL OB Protease Solution and seal the plate with Caps for Round well Plate 4 Incubate at 60 C for 10 minutes 5 Continue the protocol with Step 2 E Z 96 Tissue DNA Kit Protocols B For cells grown in a monolayer do not use more than 5 x 106 cells 1 2 3 Release the cells with trypsin Centrifuge at 300 x g for 5 minutes Resuspend cells with 200 uL cold 4 C PBS and transfer to a 96 well Round well Plate provided Add 25 uL OB Protease Solution and seal the plate with Caps for Round well Plate Incubate at 60 C for 10 minutes Continue the protocol with Step 2 2 Shake or vortex the plate vigorously from side to side do not shake up and down to avoid leaki
22. this point but does not interfere with DNA isolation Vortex for 30 seconds to mix thoroughly Note BL Buffer must be diluted with 10096 ethanol before use Please follow the instructions on Page 5 Optional Column Equilibration Protocol Place the E Z 96 DNA Plate on top of a 96 well Square well Plate provided Add 100 uL 3M NaOH into each well of the plate and let the plate sit for 4 minutes at room temperature Centrifuge at 4 000 x g for 3 minutes Discard the filtrate and reuse the 96 well Square well Plate in the next step Place the E Z 96 DNA Plate on top of a 96 well Square well Plate provided Transfer all the lysate from Step 6 to each well of the E Z 96 DNA Plate Seal the E Z 96 DNA Plate with AeraSeal film 17 10 11 12 13 14 15 16 17 18 18 E Z 96 Tissue DNA Kit Protocols Centrifuge at gt 4 000 x g for 10 minutes Note Ensure that each sample has passed through the membrane in each well ofthe E Z 96 DNA Plate Longer centrifugation may be required if any lysate remains in any of the wells If some lysate is still left in the wells even with increased centrifugation time proceed to next step Remove and discard the AeraSeal film Add 500 uL HBC Buffer to each well Seal the plate with new AeraSeal film Note HBC Buffer must be diluted with isopropanol before use Please follow the instructions on Page 5 Centrifuge at gt 4 000 x g for 5 minutes Discard the filtrate
23. tocol Steps 1 6 or Blood DNA Protocol Steps 1 8 2 Assemble the plate on the vacuum manifold according the manufacturer s instructions Optional Column Equilibration Protocol Place the E Z 96 DNA Plate on top of a 96 well Square well Plate provided Add 100 uL 3M NaOH into each well of the plate and let the plate sit for 4 minutes at room temperature Turn on the vacuum source until all the liquid passes through the E Z 96 DNA Plate 3 Transfer the lysate to the E Z 96 DNA Plate 4 Turnon the vacuum source to draw the lysate completely through the E Z 96 DNA Plate 5 Turn off the vacuum source 6 Add 500 uL HBC Buffer to each well Note HBC Buffer must be diluted with isopropanol before use Please follow the instructions on Page 5 7 Turnon the vacuum source to draw the liquid completely through the E Z 96 DNA Plate 8 Turn off the vacuum source 24 10 11 12 13 14 15 16 17 18 19 20 21 E Z 96 Tissue DNA Kit Protocols Add 600 uL DNA Wash Buffer to each well Note DNA Wash Buffer must be diluted with 10096 ethanol before use Please follow the instructions on Page 5 Turn on the vacuum source to draw the liquid completely through the E Z 96 DNA Plate Turn off the vacuum source Repeat Steps 9 11 for a second DNA Wash step Add 600 uL ethanol to each well Turn on the vacuum source to draw the liquid completely through the E Z 96 DNA Plate Continue to
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