HighPrep FFPE DNA 96. Genomic DNA isolation from FFPE (Formalin-Fix, Paraffin-Embedded) Tissue Magnetic beads based chemistry High-throughput capable

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1 MAGBIO ACCELERATING genomic research HighPrep FFPE DNA 96 Catalog Nos. HPFF-D96, HPFF-D96X4 Manual Revision v2.03 Genomic DNA isolation from FFPE (Formalin-Fix, Paraffin-Embedded) Tissue Magnetic beads based chemistry High-throughput capable PROTOCOL Contents Product Description and Process... Kit Contents, Storage, Stability... Preparation of Reagents... FFPE DNA - 96 well format... Troubleshooting guide For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. MAGBIO GENOMICS, INC. DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL MAGBIO GENOMICS, INC. BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF,WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT MAGBIO GENOMICS, INC. IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. TRADEMARKS The trademarks mentioned herein are the property of or their respective owners.

2 Product Description The Highprep FFPE DNA 96 kit is a high quality, high throughput genomic purification of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The kit is xylene-free for safer processing and ease of use. To overcome cross linking of nucleic acids caused by formalin fixation, the kit uses specially formulated buffers and lysing conditions to release DNA from tissue sections. Utilizing magnetic bead purification technology, the kit is high-throughput capable and can be adapted to most liquid handling workstations in the market. Fragmentation of Nucleic Acids Consideration The purified genomic DNA is suitable for downstream applications such as quantitative real-time RT- PCR, sequencing and mutation screening. Because tissue fixation and embedding causes significant fragmentation (nucleic acid modifications), DNA recovered from FFPE samples exhibits broad size distribution and is not recommended for downstream application that requires full length DNA. Workflow Overview The protocol is based on two methods to initially digest the FFPE sample. A non-xylene and a xylene method. The xylene approach will achieve higher yields but because xylene is toxic, it is up to the end user to determine if it is suitable for automation. The HighPrep FFPE DNA 96 kit utilizes magnetic beads technology to bind to the DNA, therefore a magnetic processing plate is required. Samples are first lysed in FFPE DTL Buffer following digestion with Pro K Solution. The lysate is then heated to denature the proteinase. FFPE DB Buffer and the MAG-S1 magnetic particles are added to bind the nucleic acid. The DNA bound beads is washed twice and eluted with MB Elution nuclease-free water. Kit Contents and Storage HighPrep FFPE DNA 96 Kits Catalog No. HPFF-D96 HPFF-D96X4 STORAGE Number of Preps FFPE DTL Buffer 30 ml 100 ml C FFPE DB Buffer 75 ml 250 ml C FFPE DW1 Buffer 1 25 ml 100 ml C FFPE DW2 Buffer 2 25 ml 100 ml C LPA 1.1 ml 4.4 ml C MB Elution Buffer 10 ml 40 ml C Pro K Solution 3 3 ml 12 ml C MAG-S1 Particles 1.1 ml 4.4 ml 2-8 C 1 Isopropanol must be added prior to use. See Preparation of Reagents 2 Ethanol must be added prior to use. See Preparation of Reagents Stability All components are stable for 12 months when stored accordingly. 3 Pro K Solution comes in a ready to use Component is stable for 1 year when stored at C. For storage longer than 1 year, store at 2-8 C is recommended. 1

3 Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs). MSDS can be downloaded from the Product Resource tab when viewing the product kit. Preparation of Reagents Prepare the following components for each kit before use: Catalog No. HPFF-D96 Component Add 100% Isopropanol FFPE DW1 Buffer 25 ml FFPE DW2 Buffer ml Add % Ethanol Storage Room Temp C Room Temp C Components are stable for 1 year when stored closed at room temperature Catalog No. HPFF-D96X4 Component Add 100% Isopropanol FFPE DW1 Buffer 100 ml FFPE DW2 Buffer ml Add % Ethanol Storage Room Temp C Room Temp C Components are stable for 1 year when stored closed at room temperature 2

4 Protocol: FFPE DNA Kit - 96 plate format Equipment and Reagents to Be Supplied by User When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs) from each product supplier. 1.2 ml or 2 ml 96 well round-bottom plates Centrifuge with swing bucket rotor for 96 deep well plate capable of 4,000 x g Water bath or heat block capable of 55 C Water bath or heat block capable of 70 C Water bath or heat block capable of 90 C Sealing film Isopropanol Ethanol (96-100%) freshly prepared. Xylene (if using xylene digestion method) Magnetic separation device for 96-well plate Things to do before starting! The protocol is based on two methods to initially digest the FFPE sample. A non-xylene and a xylene method. The xylene approach will achieve higher yields but because xylene is toxic, it is up to the end user to determine if it is suitable for automation and use. Ensure FFPE DW1 Buffer and FFPE DW2 Buffer are prepared according to the instructions on page 2 and are at room temperature. Warm up MB Elution Buffer ( 0.5 ml per sample) to 70 C Set water bath or heat block to 55 C, 70 C and 90 C. 70 C is not needed for xylene method. Protocol Choose either step 1 or step 2 of the digestion method. 1. NON XYLENE DIGESTION: a. Add 250 μl FFPE DTL Buffer to each well of the 96 well plate. b. Cut 3-8 paraffin sample sections between 5-10 μm and add to the well containing the FFPE DTL Buffer immediately. Note: Do not use the first 2-3 sections from the sample block as more paraffin is present. c. Centrifuge at 4,000 x g for 5 min. d. Seal the sample plate with a sealing film. Incubate at 70 C for 1 hour. Gently shaking the plate 2-3 times to mix during the incubation. Note: Assure the tissue samples are submerged in the 3

5 e. Tap the plate to collect the liquid from the sealing film and remove the film. Add 20 μl Pro K Solution to each sample well. Pipet mix 10 times. Seal the sample plate and incubate overnight at 55 C. Occasionally mix the plate by gently shaking the plate. Proceed to step XYLENE DIGESTION METHOD: a. Add 1 ml xylene to each well of the 96 well plate. b. Cut 3-8 paraffin sample sections between 5-10μm and add to the well containing the xylene. Note: Do not use the first 2-3 sections from the sample block as more paraffin is present. c. Seal the plate and vortex for 30 seconds. d. Centrifuge at full speed for 5 min at room temperature or until a tight pellet. e. Remove the supernatant by pipetting. Do not remove any of the pellet. f. Add 1 ml ethanol (96-100%) to the pellet, and mix by vortexing. g. Centrifuge at 4,000 g for 5 min. h. Remove the supernatant by pipetting. Do not remove any of the pellet. i. Repeat steps 2f - 2h for second ethanol wash. Remove any residual ethanol with a fine pipet tip. j. Incubate the plate at room temperature (15-25 C) or up to 37 C. Incubate for 10 min or until all residual ethanol has evaporated. k. Add 250 μl FFPE DTL Buffer and 20 μl Pro K Solution. Pipet mix 10 times. Seal the sample plate and incubate overnight at 55 C. 3. Incubate at 90 C for1 hour. Longer incubation times or higher incubation temperatures may result in more fragmented DNA. If using only one heating block, leave the sample at room temperature after the 55 C incubation until the heating block has reached 90 C. 4. Centrifuge at 4,000 x g for 5 min. The paraffin will form a thin layer on top of the lysate 5. Transfer 200 μl cleared lysate below the paraffin from each well into a new 96-well roundwell plate. Try not to transfer the paraffin material from the top layer. Suggestion: Use a 1 ml pipette tip or large orifice tip to penetrate the paraffin layer. 6. Add 500 μl FFPE DB Buffer and 10 μl MAG-S1 particles to each sample well. Pipette mix times. Note: If expecting low DNA content from sample, add 10 μl LPA (included with kit).! Shake thoroughly the MAG-S1 particles to fully resuspend before use. 4

6 7. Incubate at room temperature for 5-10 min. 8. Place the sample plate on the magnetic separation device until the beads clear the 9. Aspirate and discard the cleared supernatant. Do not disturb the magnetized beads. 10. Remove the plate from the magnetic separation device. 11. Add 400 μl FFPE DW1 Buffer to each sample well. Resuspend the beads by pipette mixing times. (See Preparation of Reagents on page 2 for FFPE DW1 Buffer) 12. Place the sample plate on the magnetic separation device until the beads clear the 13. Aspirate and discard the cleared supernatant. Do not disturb the magnetized beads. 14. Remove the plate from the magnetic separation device. 15. Add 400 μl FFPE DW2 Buffer to each sample well. Resuspend the beads by pipette mixing times. (See Preparation of Reagents on page 2 for FFPE DW2 Buffer) 16. Place the sample plate on the magnetic separation device until the beads clear the 17. Aspirate and discard the cleared supernatant. Do not disturb the magnetized beads. 18. Repeat steps for a second FFPE DW2 Buffer wash. 19. Leave the plate on the magnetic separation device for 5-10 min to air dry the magnetized beads. It is critical to completely remove all liquid from each well since ethanol carryover in the eluate may interfere with some downstream applications. Do not over dry the beads. 20. Add μl MB Elution Buffer to each sample well. Resuspend the beads by pipette mixing 30 times. Alternatively, nuclease-free water may be used. 21. Let the sample plate Incubate at room temperature for 10 min. 22. Place the sample plate on the magnetic separation device until the beads clear the 23. Pipet the cleared supernatant containing purified DNA to a new storage microplate. Store the DNA at -20 C. 5

7 Troubleshooting guide Please use this guide to troubleshoot any problems that may arise. For further assistance, please contact technical support via: Phone: (within US), outside US, Symptoms Possible Causes Comments Low DNA Yields MAG-S1 particles do not completely clear from solution Tissue was not digested. Incomplete resuspension of MAG-S1 particles. Loss of MAG-S1 particles during operation. FFPE DW1 Buffer and FFPE DW2 Buffer were not prepared correctly. Binding was incomplete. Too short of magnetizing time. Ensure that the tissue is fully submerged during the digestion steps. Resuspend MAG-S1 particles by pipette mixing or vortexing vigorously before use. Avoid disturbing the MAG-S1 particles during aspiration of supernatant. Prepare buffers accordingly. See Preparation of Reagents on page 2. Ensure that the samples are mixed well before collecting the beads. Increase pipette mixing or vortex/shake plate to ensure complete mixing. Increase collection time on the magnet. Problems in downstream applications Carryover of the magnetic beads in the elution DNA is over fixated during tissue formalin fixation. Bead collection time is too short. Extend incubation time at 90 C to 90 min. Increase bead collection time. Residual magnetic particles in eluted DNA will not affect downstread application. Residual magnetic particles in eluted DNA can be magnetized again and the DNA eluate can be transfered to a new storage plate. Ordering Information Product Description Catalog No. Preps HighPrep FFPE DNA 96 HPFF-D96 96 HighPrep FFPE DNA 96 (96 x 4) HPFF-D96X

8 MAGBIO ACCELERATING genomic research