E.Z.N.A.® FFPE DNA Kit - Omega Bio-Tek
Contents
1. Aspirate and discard the supernatant Do not disturb the pellet With the lid open dry the pellet at 37 C for 15 minutes Carefully aspirate any residual ethanol with a pipettor before proceeding to the next step Add 200 uL FTL Buffer and pipette up and down to resuspend the pellet Add 20 uL Proteinase K Solution and vortex to mix thoroughly Incubate at 55 C for 3 hours Note Incubation can proceed overnight Incubate at 90 C for 10 30 minutes Centrifuge the tube briefly to collect any liquid adhering to the lid Optional If RNA free gDNA is required add 10 uL RNase A 20 mg mL not provided and incubate for 5 minutes at room temperature 16 17 18 Add 220 uL BL Buffer Vortex to mix thoroughly Add 250 uL 100 ethanol Vortex to mix thoroughly Place a MicroElute DNA Mini Column in a 2 mL Collection Tube provided with this kit E Z N A FFPE DNA Kit Protocols Optional Protocol for Column Equilibration 19 20 21 22 23 24 25 26 27 28 1 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column 2 Centrifuge at maximum speed for 30 60 seconds 3 Discard the filtrate and reuse the collection tube Transfer the entire sample from Step 17 including any precipitate that may have formed to the MicroElute DNA Mini Column Centrifuge at 10 000 x g for 1 minute at room temperature to bind the DNA to the column membrane Discard the filtrate and the collec2. C E Z N A FFPE DNA Kit Protocols E Z N A FFPE DNA Kit Protocol Heat Extraction Method Materials and Equipment to be Supplied by User 100 Ethanol Isopropanol Centrifuge capable of 14 000 x g 1 5 mL or 2 mL nuclease free microcentrifuge tubes Nuclease free pipette tips Water baths or heat blocks capable of 55 C 70 C and 90 C Optional RNase A 20 mg mL Optional 3M NaOH Before Starting Heat the water bath or heat block to 55 C Heat the water bath or heat block to 90 C Heat Elution Buffer to 70 C for the elution step Prepare the DNA Wash Buffer and HBC Buffer according to the instructions in the Preparing Reagents section on Page 4 Note All centrifugation steps must be performed at room temperature 1 Add 200 uL FTL Buffer into a 1 5 mL or 2 mL microcentrifuge tube not provided Cut 3 4 paraffin sections 5 10 um thick Note Do not use the first 2 3 sections Immediately place the section s into the tube containing FTL Buffer Vortex for 20 seconds to mix thoroughly Incubate at 90 C for 15 minutes to melt the paraffin Mix the sample a few times by gently shaking the tube 2 3 times Make sure that the tissue sections stay submerged in the solution E Z N A FFPE DNA Kit Protocols 6 Leave sample at room temperature for 5 minutes to allow to cool before adding Proteinase K Solution Note If the sample temperature is too high Proteinase K can be inactivated 7 Add 20 uL Prot
3. A 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 provided with the kit Centrifuge at full speed 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 Place the MicroElute DNA Mini Column into a new 1 5 mL microcentrifuge tube not provided Add 50 75 uL Elution Buffer heated to 70 C directly to the center of the column membrane Let sit for 3 minutes at room temperature Centrifuge at maximum speed for 1 minute to elute DNA Repeat Steps 16 18 for a second elution step Store eluted DNA at 20 C 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 800 832 8896 Incomplete cell lysis or Increase incubation time with FTL Buffer and crosslinking removal protease Ensure that no visible pieces of tissue remain Samples are rich in After applying to column wash with 300 uL protein of a 1 1 mixture of BL Buffer and ethanol and then with DNA Wash Buffer Poor cell lysis due to Mix thoroughly with BL Buffer prior to loading improper mixing with the MicroElute DNA Mini Column BL Buffer Poor cell and or pro Tissue sample must be cut or min
4. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z N A FFPE DNA Kit D3399 00 5 preps D3399 01 50 preps June 2013 For research use only Not intended for diagnostic testing E Z N A FFPE DNA Kit Table of Contents Introduction and OVErVIEW scssssssccssesseccseecseesscessecseecneceees Kit Contents Storage and Stability sesseesssececsseeses Preparing Reagents esssesssssssseseeesssesesessssesceesseseesoesesssoosssseee FFPE DNA Kit Protocol Xylene Extraction sessceeccneen FFPE DNA Kit Protocol Heat Extraction Supplemental Vacuum ProtoCol ssssssessessssssssssesseesssssssssssee Troubleshooting Guide sesserserserseseresesresrssreerrerserssssssssess Manual Revision June 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction E Z N A FFPE DNA Kit provides a rapid and easy method for the isolation of genomic DNA from FFPE tissue sections There is no need for phenol chloroform extraction and time consuming steps such as precipitation with isopropanol or ethanol are eliminated DNA purified using the E Z N A FFPE DNA method is ready for applications such as PCR Overview E Z N A FFPE DNA Kit combines MicroElute DNA Mini Column technology with a proprietary buffer system to provide a fast and easy method for DNA isolation from FFPE samples The sample is heat treated with FTL Buffer f
5. action method can be used to remove the paraffin prior to DNA extraction via the vacuum method Carry out deparaffinization Proteinase K digestion and column equilibration as indicated in either of the two preceding protocols Instead of continuing with the initial sample transfer to the MicroElute DNA Mini Column Step 22 in the Xylene Method Page 7 or Step 16 in the Heat Method Page 10 follow the steps below Note Please read through the preceding protocols of this manual before using this protocol 1 Prepare the vacuum manifold according to manufacturer s instructions 2 Connect the MicroElute DNA Mini Column to the vacuum manifold 3 Load the sample from Step 17 in the Xylene Method Page 6 or Step 11 in the Heat Method Page 10 onto MicroElute DNA Mini Column 4 Turn on vacuum source to draw the sample through the column 5 Turn off the vacuum 6 Add 500 uL HBC Buffer to the MicroElute DNA Mini Column Note HBC Buffer must be diluted with isopropanol before use Please see Page 4 for instructions 7 Turn on vacuum source to draw the HBC Buffer through the column 13 10 11 12 13 14 13 16 17 18 19 20 14 E Z N A FFPE DNA Kit Protocols Turn off the vacuum Add 700 uL DNA Wash Buffer to the MicroElute DNA Mini Column Note DNA Wash Buffer must be diluted with ethanol before use Please see Page 4 for instructions Turn on vacuum source to draw the DN
6. ced into tein lysis in small pieces Increase incubation time at FTL Buffer 55 C with FTL Buffer to ensure that tissue is completely lysed Ethanol not added to Dilute DNA Wash Buffer with the indicated DNA Wash Buffer volume of 100 ethanol before use lsopropanol not Dilute HBC Wash Buffer with the indicated added to HBC Buffer volume of isopropanol before use Washing Incomplete lysis due BL Buffer is viscous and the sample must be leaves to improper mixing vortexed thoroughly colored with BL Buffer residuein Ethanol not added to Dilute DNA Wash Buffer with the indicated column DNA Wash Buffer volume of 100 ethanol before use HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 15 Notes
7. einase K Solution and vortex to mix thoroughly 8 Incubate at 55 C for 3 hours Note Incubation can proceed overnight 9 Centrifuge the tube briefly to collect any liquid adhering to the lid Optional If RNA free gDNA is required add 10 uL RNase A 20 mg mL not provided and incubate for 5 minutes at room temperature 10 Add 220 uL BL Buffer and vortex to mix thoroughly 11 Add 250 uL 100 ethanol and vortex to mix thoroughly 12 Place a MicroElute DNA Mini Column in a 2 mL Collection Tube provided with this kit Optional Protocol for Column Equilibration 1 Add 100 uL 3M NaOH to the MicroElute DNA Mini Column 2 Centrifuge at maximum speed for 30 60 seconds 3 Discard the filtrate and reuse the collection tube 13 Transfer the entire sample from Step 11 including any precipitate that may have formed to the MicroElute DNA Mini Column 10 14 15 16 17 18 19 20 21 22 23 24 25 E Z N A FFPE DNA Kit Protocols Centrifuge at 10 000 x g for 1 minute at room temperature to bind the DNA to the column membrane Discard the filtrate and the collection tube Transfer the MicroElute DNA Mini Column to a new 2 mL Collection Tube provided with this kit Add 500 uL HBC Buffer to the MicroElute DNA Mini Column 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 at room temperat
8. ilute HBC Buffer with isopropanol as follows and store at room temperature E Z N A FFPE DNA Kit Protocols E Z N A FFPE DNA Kit Protocol Xylene Extraction Method Materials and Equipment to be Supplied by User 100 Ethanol e lsopropanol e Xylene Microcentrifuge capable of 14 000 x g e 1 5 mL or 2 mL nuclease free microcentrifuge tubes e Nuclease free pipette tips Water baths or heat blocks capable of 37 C 55 C 70 C and 90 C Optional RNase A 20 mg mL e Optional 3M NaOH Before Starting Heat the water bath or heat block to 37 C Heat the water bath or heat block to 55 C Heat the water bath or heat block to 90 C Heat Elution Buffer to 70 C for the elution step Prepare the DNA Wash Buffer and HBC Buffer according to the instructions in the Preparing Reagents section on Page 4 1 Add 1 mL xylene toa 1 5 mL or 2 mL microcentrifuge tube not provided 2 Cut 3 8 paraffin sections 5 10 um thick Note Do not use the first 2 3 sections 3 Immediately place the section s into the tube containing xylene 4 Vortex for 20 seconds to mix thoroughly 5 Centrifuge at maximum speed for 2 minutes at room temperature 6 Aspirate and discard the supernatant Do not disturb the pellet 10 11 12 13 14 15 E Z N A FFPE DNA Kit Protocols Add 1 mL 100 ethanol to the tube and vortex to mix thoroughly Centrifuge at maximum speed for 2 minutes at room temperature
9. ollowed by Proteinase K digestion to release DNA After adjusting the binding conditions with ethanol the lysate is applied to the MicroElute DNA Mini Column to bind DNA Cellular debris and proteins are effectively washed away during the wash steps High quality DNA is eluted in sterile deionized water or low salt buffer Binding Capacity Each MicroElute DNA Mini Column can bind approximately 100 ug DNA Using greater than 30 mg FFPE tissue 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 is no longer included with this kit An optional Column Equilibration Protocol has been added to the protocol for your convenience e Equilibration Buffer is replaced with 3M NaOH provided by the user Kit Contents reine T oo oo Storage and Stability All components of the E Z N A FFPE DNA Kit can be stored at room temperature and are guaranteed for at least 12 months from the date of purchase Proteinase K Solution can be stored at room temperature for 12 months For long term storage gt 12 months store Proteinase K Solution at 2 8 C Under cool ambient conditions a precipitate may form in the BL Buffer In case of such an event heat the bottle at 37 C to dissolve Store BL Buffer at room temperature Preparing Reagents 1 Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature 2 D
10. tion tube Transfer the MicroElute DNA Mini Column to a new 2 mL Collection Tube provided with this kit Add 500 uL HBC Buffer to the MicroElute DNA Mini Column 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 at room temperature Discard the filtrate and the collection tube Transfer the MicroElute DNA Mini Column to a new 2 mL Collection Tube provided with this kit Add 700 uL DNA Wash Buffer to the MicroElute DNA Mini Column Note DNA Wash Buffer must be diluted with ethanol before use Please see Page 4 for instructions Centrifuge at 10 000 x g for 1 minute at room temperature 29 30 31 32 33 34 35 36 37 E Z N A FFPE DNA Kit Protocols Discard the filtrate and reuse the collection tube Repeat Steps 27 29 for a second DNA Wash step Centrifuge at full speed 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 Place the MicroElute DNA Mini Column into a new 1 5 mL microcentrifuge tube not provided Add 50 75 uL Elution Buffer heated to 70 C directly to the center of the column membrane Let sit for 3 minutes at room temperature Centrifuge at maximum speed for 1 minute to elute DNA Repeat Steps 33 35 for a second elution step Store eluted DNA at 20
11. ure Discard the filtrate and the collection tube Transfer the MicroElute DNA Mini Column to a new 2 mL Collection Tube provided with this kit Add 700 uL DNA Wash Buffer to the MicroElute DNA Mini Column Note DNA Wash Buffer must be diluted with ethanol before use Please see Page 4 for instructions Centrifuge at 10 000 x g for 1 minute at room temperature Discard the filtrate and reuse the collection tube Repeat Steps 21 23 for a second DNA Wash step Centrifuge at full speed for 3 minutes to completely dry the membrane Note It is important to dry the column membrane before elution Residual ethanol may interfere with downstream applications 11 26 27 28 29 30 31 12 E Z N A FFPE DNA Kit Protocols Place the MicroElute DNA Mini Column into a new 1 5 mL microcentrifuge tube not provided Add 50 75 uL Elution Buffer heated to 70 C directly to the center of the column membrane Let sit for 3 minutes at room temperature Centrifuge at maximum speed for 1 minute to elute DNA Repeat Steps 27 29 for a second elution step Store eluted DNA at 20 C E Z N A FFPE DNA Kit Protocols E Z N A FFPE DNA Kit Protocol Vacuum Method Materials and Equipment to be Supplied by User Vacuum manifold Cat VAC 08 e Vacuum source or pump The vacuum method outlined here is an alternative to centrifugation steps presented in the protocols above Either the xylene or heat extr
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