E.Z.N.A. Size Select-IT Kit D6488-00 10 preps D6488-01 50 preps D6488-02 200 preps September 2012
E.Z.N.A. Size Select-IT Table of Contents Introduction...2 Illustrated Protocol...3 Size Selection Guide...4 Kit Contents/Storage and Stability...7 Preparing Reagents...8 Recovery of Fragments 150-500 bp Protocol...9 Recovery of Fragments 150-400 bp Protocol...12 Recovery of Fragments 150-300 bp Protocol...15 Recovery of Fragments 150-700 bp Protocol...18 Troubleshooting...21 Ordering...22 Manual Revision: September 2012 Innovations in nucleic acid isolation 1
Introduction Post DNA shearing, the library construction process for next generation sequencing (NGS) requires fragment size selection regardless of the sequencer platform. Obtaining a high recovery rate and a precise DNA size distribution range is key to reduce sequencing bias. The E.Z.N.A. Size Select-IT Kit is optimized for use with the library construction workflows for all the common NGS platforms such as Illumina HiSeq/GA/MiSeq, Roche 454 Genome Sequencer and Life Technologies SOLiD/Ion Torrent. The E.Z.N.A. Size Select-IT Kit utilizes a unique spin column based technology for quick and more optimized DNA size selection suitable for various next generation sequencing platforms. The E.Z.N.A. Size Select-IT Kit is a convenient system for fast and reliable processing of library size selection and purification of enzymatic reactions. The E.Z.N.A. Size Select-IT Kit can selectively and precisely recover DNA fragments between 150 bp to 700 bp free of oligonucleotides, nucleotides, and polymerase with recovery rate exceeding 50%. DNA binding condition is adjusted by adding appropriate volumes of the specially formulated buffers before loading to the column. Following two rapid wash steps, DNA can be eluted with deionized water or a low salt buffer. Benefits of the E.Z.N.A. Size Select-IT Kit Simplicity - No tedious gel purification, no specialize equipment to buy Efficiency - DNA recovery from enzymatic reactions can be achieved in less than 10 minutes Flexibility - Customizable protocol to fine-tune DNA recovery size range Reliability - Optimized buffers that guarantee pure DNA Binding Capacity Each HiBind NGS DNA Mini Columns can bind ~5 µg DNA. New In this Edition The HiBind NGS DNA Mini Columns have been redesigned to increase DNA recovery by reducing the HiBind matrix retention volume. 2
Illustrated Protocol Add XE1 Binding Buffer and ACD Buffer Transfer to NGS LS DNA Mini Column Add XE2 Buffer and BCS Buffer to the filtrate Transfer to NGS SS DNA Mini Column Wash 2x Dry Elute 3
Size Selection Guide E.Z.N.A. Size Select-IT Kit Size Selection Guides The E.Z.N.A. DNA Size Select-IT Kit offers precise DNA size selection by adding various volumes of ACD Buffer and/or BCS Buffer to the DNA fragment sample. The standard protocols in this booklet are designed for DNA samples dissolved in water or common elution buffers such as Omega Bio-tek s Elution Buffer or Qiagen s EB Buffer. However, DNA size selection range will vary depending on the salt concentration and ph of the DNA sample. If using DNA in buffer other than aforementioned, it is highly advised that one should perform a titration test to confirm the cut-off range of DNA size distribution by using different volumes of ACD Buffer (removal of large DNA fragments) and BCS Buffer (removal of small DNA fragments). Below is a brief outline of the titration test procedure. Please refer to the main manual for procedural details. Removal of Large DNA Fragments The E.Z.N.A. DNA Size Select-IT Kit protocols can be adjusted to fit each user s different DNA fragment size requirements. ACD Buffer selectively removes larger bp fragments from a sheared DNA sample. By increasing the volume of ACD Buffer added to the sheared DNA sample, the larger DNA size cut-off decreases. For example, if DNA is in water or common elution buffer, and fragments less than 400 bp are required, add 8 μl ACD Buffer to the sheared DNA sample (please refer to the table below). 1. Mix 50 µl DNA, 200 µl XE1 Binding Buffer, and various volumes of ACD Buffer (6-14 µl as a guideline. See table below and the Bioanalyzer traces on Page 5 for details.) 2. Pass through the NGS LS DNA Mini Column. 3. To the filtrate add 1 volume XE2 Buffer. 4. Pass through the NGS SS DNA Mini Column. 5. Wash the NGS SS DNA Mini Column twice with DNA Wash Buffer. 6. Spin dry the column. 7. Elute with 20-30 µl Elution Buffer. 8. Perform gel or Bioanalyzer analysis to determine the optimal volume of ACD Buffer needed for the desired large DNA size cut-off. ACD Buffer 6 μl 8 μl 10 μl 12 μl 14 μl DNA Size Cut-off 500 bp 400 bp 300 bp 250 bp 0 bp 4
Size Selection Guide 6 μl ACD Buffer 8 μl ACD Buffer 10 μl ACD Buffer 500 bp 400 bp 300 bp 12 μl ACD Buffer 14 μl ACD Buffer 250 bp Removal of Small DNA Fragments The E.Z.N.A. DNA Size Select-IT Kit protocols can be adjusted to fit each user s different DNA fragment size requirements. BCS Buffer selectively removes smaller bp fragments from a sheared DNA sample. By increasing the volume of BCS Buffer added to the sheared DNA sample, the smaller DNA size cut-off increases. For example, if DNA is in water or common elution buffer, and fragments greater than 150 bp are required, add 20 μl BCS Buffer to the sheared DNA sample (please refer to the table on Page 6). 1. Mix 50 µl DNA, 200 µl XE1 Binding Buffer, and the optimal volume of ACD Buffer as determined in the previous section. 2. Pass through the NGS LS DNA Mini Column. 3. To the filtrate add 1 volume XE2 Buffer and various volumes of BCS Buffer (10-20 µl as a guideline. See table and Bioanalyzer traces on Page 6 for details.). 4. Pass through the NGS SS DNA Mini Column. 5. Wash the NGS SS DNA Mini Column twice with DNA Wash Buffer. 6. Spin dry the column. 7. Elute with 20-30 µl Elution Buffer. 8. Perform gel or Bioanalyzer analysis to determine the optimal volume of BCS Buffer needed for the desired small DNA size cut-off. 5
Size Selection Guide BCS Buffer (μl) Low Cut-off Size Range (bp) ACD Buffer (μl) High Cut-off 4-10 100-500 6 12 110-500 6 14 120-500 6 16 130-500 6 18 140-500 6 20 150-700 3 20 150-500 6 20 150-400 8 20 150-300 10 20 150-250 12 10 μl BCS Buffer 6 μl ACD Buffer 12 μl BCS Buffer 6 μl ACD Buffer 14 μl BCS Buffer 6 μl ACD Buffer 100 bp 500 bp 110 bp 500 bp 120 bp 500 bp 16 μl BCS Buffer 6 μl ACD Buffer 18 μl BCS Buffer 6 μl ACD Buffer 20 μl BCS Buffer 6 μl ACD Buffer 130 bp 500 bp 140 bp 500 bp 150 bp 500 bp 6
Kit Contents Size Select-IT Kit D6488-00 D6488-01 D6488-02 Preps 10 50 200 NGS LS DNA Mini Column 10 50 200 NGS SS DNA Mini Column 10 50 200 2 ml Collection Tubes 20 100 400 XE1 Binding Buffer 3 ml 12 ml 50 ml XE2 Buffer 3 ml 12 ml 50 ml ACD Buffer 200 μl 1 ml 4 ml BCS Buffer 300 μl 1.5 ml 5 ml DNA Wash Buffer 2 x 1.5 ml 15 ml 2 x 25 ml Elution Buffer 5 ml 5 ml 15 ml User Manual P P P Storage and Stability All E.Z.N.A. Size Select-IT Purification Kit components are guaranteed for at least 12 months from the date of purchase when stored at 22-25 C. If any precipitates form in buffers, warm at 37 C to dissolve. 7
Preparing Reagents Dilute DNA Wash Buffer with 100% ethanol as follows and store at room temperature. Kit Ethanol To Be Added D6488-00 6 ml per bottle D6488-01 60 ml D6488-02 100 ml per bottle 8
E.Z.N.A. Size Select-IT Kit Protocol - Recovery of 150-500 bp Fragments The protocol below is validated using DNA dissolved in water or common elution buffers such as Omega Bio-tek s Elution Buffer or Qiagen s EB Buffer. However, DNA size selection range will vary depending on the salt concentration and ph of the DNA sample. If using DNA in buffer other than aforementioned, or the desired size range is not listed in this manual, it is highly advised that one should perform a titration test to confirm the cut-off range of DNA size distribution by using different volumes of ACD Buffer (removal of large DNA fragments) and BCS Buffer (removal of small DNA fragments). Please refer to the previous section (Size Selection Guide) for titration test procedure. Materials and Equipment to be Supplied by User: Microcentrifuge capable of at least 13,000 x g Nuclease-free 1.5 ml microcentrifuge tubes 100% ethanol Optional: Sterile deionized water or TE Buffer Before Starting: Prepare DNA Wash Buffer according to Preparing Reagents section on Page 8. Prepare sheared DNA and verify the fragments by gel electrophoresis. 1. Add 50 μl DNA fragment dissolved in sterile deionized water or TE Buffer to a nuclease-free 1.5 ml microcentrifuge tube. Note: If less than 50 μl sheared DNA is available, bring the volume up to 50 μl with sterile deionized water or TE Buffer. 2. Add 200 μl XE1 Binding Buffer and 6 μl ACD Buffer. Vortex to mix thoroughly. 3. Centrifuge briefly to collect any droplets from the inside of the lid. 4. Insert a NGS LS DNA Mini Column into a 2 ml Collection Tube. 5. Transfer the sample to the NGS LS DNA Mini Column. 9
6. Centrifuge at 13,000 x g for 30 seconds at room temperature. 7. Discard the NGS LS DNA Mini Column and transfer the filtrate to a clean 1.5 ml microcentrifuge tube. 8. Add 1 volume XE2 Buffer (with respect to the filtrate volume). For example, if the filtrate volume is 200 µl, add 200 µl XE2 Buffer. 9. Add 20 µl BCS Buffer. Vortex to mix thoroughly. 10. Centrifuge briefly to collect any droplets from the inside of the lid. 11. Insert a NGS SS DNA Mini Column into a 2 ml Collection Tube. 12. Transfer the sample to the NGS SS DNA Mini Column. 13. Centrifuge at 13,000 x g for 30 seconds at room temperature. 14. Discard the filtrate and reuse the collection tube. 15. Add 700 µl DNA Wash Buffer. Note: DNA Wash Buffer must be diluted with ethanol prior to use. Please see Page 8 for instructions. 16. Centrifuge at 13,000 x g for 30 seconds. 17. Discard the filtrate and reuse the collection tube. 18. Add 500 µl DNA Wash Buffer. 19. Centrifuge at 13,000 x g for 30 seconds. 10
20. Discard the filtrate and reuse the collection tube. 21. Centrifuge the empty NGS SS DNA Mini Column for 2 minutes at maximum speed ( 13,000 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. 22. Transfer the NGS SS DNA Mini Column into a clean 1.5 ml microcentrifuge tube. 23. Add 20-30 µl Elution Buffer, sterile deionized water, or TE Buffer directly onto the center of column matrix. 24. Let it sit at room temperature for 2 minutes. 25. Centrifuge at 13,000 x g for 1 minute. 26. Store eluted DNA at -20 C. 11
E.Z.N.A. Size Select-IT Kit Protocol - Recovery of 150-400 bp Fragments The protocol below is validated using DNA dissolved in water or common elution buffers such as Omega Bio-tek s Elution Buffer or Qiagen s EB Buffer. However, DNA size selection range will vary depending on the salt concentration and ph of the DNA sample. If using DNA in buffer other than aforementioned, or the desired size range is not listed in this manual, it is highly advised that one should perform a titration test to confirm the cut-off range of DNA size distribution by using different volumes of ACD Buffer (removal of large DNA fragments) and BCS Buffer (removal of small DNA fragments). Please refer to the previous section (Size Selection Guide) for titration test procedure. Materials and Equipment to be Supplied by User: Microcentrifuge capable of at least 13,000 x g Nuclease-free 1.5 ml microcentrifuge tubes 100% ethanol Optional: Sterile deionized water or TE Buffer Before Starting: Prepare DNA Wash Buffer according to Preparing Reagents section on Page 8. Prepare sheared DNA and verify the fragments by gel electrophoresis. 1. Add 50 μl DNA fragment dissolved in sterile deionized water or TE Buffer to a nuclease-free 1.5 ml microcentrifuge tube. Note: If less than 50 μl sheared DNA is available, bring the volume up to 50 μl with sterile deionized water or TE Buffer. 2. Add 200 μl XE1 Binding Buffer and 8 μl ACD Buffer. Vortex to mix thoroughly. 3. Centrifuge briefly to collect any droplets from the inside of the lid. 4. Insert a NGS LS DNA Mini Column into a 2 ml Collection Tube. 12
5. Transfer the sample to the NGS LS DNA Mini Column. 6. Centrifuge at 13,000 x g for 30 seconds at room temperature. 7. Discard the NGS LS DNA Mini Column and transfer the filtrate to a clean 1.5 ml microcentrifuge tube. 8. Add 1 volume XE2 Buffer (with respect to the filtrate volume). For example, if the filtrate volume is 200 µl, add 200 µl XE2 Buffer. 9. Add 20 µl BCS Buffer. Vortex to mix thoroughly. 10. Centrifuge briefly to collect any droplets from the inside of the lid. 11. Insert a NGS SS DNA Mini Column into a 2 ml Collection Tube. 12. Transfer the sample to the NGS SS DNA Mini Column. 13. Centrifuge at 13,000 x g for 30 seconds at room temperature. 14. Discard the filtrate and reuse the collection tube. 15. Add 700 µl DNA Wash Buffer. Note: DNA Wash Buffer must be diluted with ethanol prior to use. Please see Page 8 for instructions. 16. Centrifuge at 13,000 x g for 30 seconds. 17. Discard the filtrate and reuse the collection tube. 18. Add 500 µl DNA Wash Buffer. 13
19. Centrifuge at 13,000 x g for 30 seconds. 20. Discard the filtrate and reuse the collection tube. 21. Centrifuge the empty NGS SS DNA Mini Column for 2 minutes at maximum speed ( 13,000 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. 22. Transfer the NGS SS DNA Mini Column into a clean 1.5 ml microcentrifuge tube. 23. Add 20-30 µl Elution Buffer, sterile deionized water, or TE Buffer directly onto the center of column matrix. 24. Let it sit at room temperature for 2 minutes. 25. Centrifuge at 13,000 x g for 1 minute. 26. Store eluted DNA at -20 C. 14
E.Z.N.A. Size Select-IT Kit Protocol - Recovery of 150-300 bp Fragments The protocol below is validated using DNA dissolved in water or common elution buffers such as Omega Bio-tek s Elution Buffer or Qiagen s EB Buffer. However, DNA size selection range will vary depending on the salt concentration and ph of the DNA sample. If using DNA in buffer other than aforementioned, or the desired size range is not listed in this manual, it is highly advised that one should perform a titration test to confirm the cut-off range of DNA size distribution by using different volumes of ACD Buffer (removal of large DNA fragments) and BCS Buffer (removal of small DNA fragments). Please refer to the previous section (Size Selection Guide) for titration test procedure. Materials and Equipment to be Supplied by User: Microcentrifuge capable of at least 13,000 x g Nuclease-free 1.5 ml microcentrifuge tubes 100% ethanol Optional: Sterile deionized water or TE Buffer Before Starting: Prepare DNA Wash Buffer according to Preparing Reagents section on Page 8. Prepare sheared DNA and verify the fragments by gel electrophoresis. 1. Add 50 μl DNA fragment dissolved in sterile deionized water or TE Buffer to a nuclease-free 1.5 ml microcentrifuge tube. Note: If less than 50 μl sheared DNA is available, bring the volume up to 50 μl with sterile deionized water or TE Buffer. 2. Add 200 μl XE1 Binding Buffer and 10 μl ACD Buffer. Vortex to mix thoroughly. 3. Centrifuge briefly to collect any droplets from the inside of the lid. 4. Insert a NGS LS DNA Mini Column into a 2 ml Collection Tube. 15
5. Transfer the sample to the NGS LS DNA Mini Column. 6. Centrifuge at 13,000 x g for 30 seconds at room temperature. 7. Discard the NGS LS DNA Mini Column and transfer the filtrate to a clean 1.5 ml microcentrifuge tube. 8. Add 1 volume XE2 Buffer (with respect to the filtrate volume). For example, if the filtrate volume is 200 µl, add 200 µl XE2 Buffer. 9. Add 20 µl BCS Buffer. Vortex to mix thoroughly. 10. Centrifuge briefly to collect any droplets from the inside of the lid. 11. Insert a NGS SS DNA Mini Column into a 2 ml Collection Tube. 12. Transfer the sample to the NGS SS DNA Mini Column. 13. Centrifuge at 13,000 x g for 30 seconds at room temperature. 14. Discard the filtrate and reuse the collection tube. 15. Add 700 µl DNA Wash Buffer. Note: DNA Wash Buffer must be diluted with ethanol prior to use. Please see Page 8 for instructions. 16. Centrifuge at 13,000 x g for 30 seconds. 17. Discard the filtrate and reuse the collection tube. 18. Add 500 µl DNA Wash Buffer. 16
19. Centrifuge at 13,000 x g for 30 seconds. 20. Discard the filtrate and reuse the collection tube. 21. Centrifuge the empty NGS SS DNA Mini Column for 2 minutes at maximum speed ( 13,000 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. 22. Transfer the NGS SS DNA Mini Column into a clean 1.5 ml microcentrifuge tube. 23. Add 20-30 µl Elution Buffer, sterile deionized water, or TE Buffer directly onto the center of column matrix. 24. Let it sit at room temperature for 2 minutes. 25. Centrifuge at 13,000 x g for 1 minute. 26. Store eluted DNA at -20 C. 17
E.Z.N.A. Size Select-IT Kit Protocol - Recovery of 150-700 bp Fragments The protocol below is validated using DNA dissolved in water or common elution buffers such as Omega Bio-tek s Elution Buffer or Qiagen s EB Buffer. However, DNA size selection range will vary depending on the salt concentration and ph of the DNA sample. If using DNA in buffer other than aforementioned, or the desired size range is not listed in this manual, it is highly advised that one should perform a titration test to confirm the cut-off range of DNA size distribution by using different volumes of ACD Buffer (removal of large DNA fragments) and BCS Buffer (removal of small DNA fragments). Please refer to the previous section (Size Selection Guide) for titration test procedure. Materials and Equipment to be Supplied by User: Microcentrifuge capable of at least 13,000 x g Nuclease-free 1.5 ml microcentrifuge tubes 100% ethanol Optional: Sterile deionized water or TE Buffer Before Starting: Prepare DNA Wash Buffer according to Preparing Reagents section on Page 8. Prepare sheared DNA and verify the fragments by gel electrophoresis. 1. Add 50 μl DNA fragment dissolved in sterile deionized water or TE Buffer to a nuclease-free 1.5 ml microcentrifuge tube. Note: If less than 50 μl sheared DNA is available, bring the volume up to 50 μl with sterile deionized water or TE Buffer. 2. Add 200 μl XE1 Binding Buffer and 3 μl ACD Buffer. Vortex to mix thoroughly. 3. Centrifuge briefly to collect any droplets from the inside of the lid. 4. Insert a NGS LS DNA Mini Column into a 2 ml Collection Tube. 18
5. Transfer the sample to the NGS LS DNA Mini Column. 6. Centrifuge at 13,000 x g for 30 seconds at room temperature. 7. Discard the NGS LS DNA Mini Column and transfer the filtrate to a clean 1.5 ml microcentrifuge tube. 8. Add 1 volume XE2 Buffer (with respect to the filtrate volume). For example, if the filtrate volume is 200 µl, add 200 µl XE2 Buffer. 9. Add 20 µl BCS Buffer. Vortex to mix thoroughly. 10. Centrifuge briefly to collect any droplets from the inside of the lid. 11. Insert a NGS SS DNA Mini Column into a 2 ml Collection Tube. 12. Transfer the sample to the NGS SS DNA Mini Column. 13. Centrifuge at 13,000 x g for 30 seconds at room temperature. 14. Discard the filtrate and reuse the collection tube. 15. Add 700 µl DNA Wash Buffer. Note: DNA Wash Buffer must be diluted with ethanol prior to use. Please see Page 8 for instructions. 16. Centrifuge at 13,000 x g for 30 seconds. 17. Discard the filtrate and reuse the collection tube. 18. Add 500 µl DNA Wash Buffer. 19
19. Centrifuge at 13,000 x g for 30 seconds. 20. Discard the filtrate and reuse the collection tube. 21. Centrifuge the empty NGS SS DNA Mini Column for 2 minutes at maximum speed ( 13,000 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. 22. Transfer the NGS SS DNA Mini Column into a clean 1.5 ml microcentrifuge tube. 23. Add 20-30 µl Elution Buffer, sterile deionized water, or TE Buffer directly onto the center of column matrix. 24. Let it sit at room temperature for 2 minutes. 25. Centrifuge at 13,000 x g for 1 minute. 26. Store eluted DNA at -20 C. 20
Troubleshooting Guide Please use this guide to troubleshot any problems that may arise. For further assistance, please contact the technical support staff, toll free, at 1-800-832-8896. Possible Problems and Suggestions Low DNA yields Elution Using Water: Water ph is too low (< 7.5 ) Check the ph of the water, adjust the ph of the water to 8.0 using Tris-HCl (2M, ph 8.5) No DNA eluted DNA Wash Buffer has not been diluted with 100% ethanol Prepare DNA Wash Buffer as instructed on the bottle, or refer to the Preparing Reagents section on Page 8. Optical densities do not agree with DNA yield on agarose gel Trace contaminants eluted from column increase A260. Make sure to wash column as instructed in Steps 15 and 19 of any protocol, rely on agarose gel/ethidium bromide electrophoresis for quantization. DNA sample floats out of well while loading agarose gel Ethanol not completely removed from column. Centrifuge the empty as instructed in Step 21 of any protocol. 21
Ordering Information The following components are available for purchase separately. (Call Toll-free at 1-800-832-8896) Product Part Number Elution Buffer, 100 ml PDR048 DNA Wash Buffer, 100 ml PS010 2 ml Collection Tubes SS1-1370-00 1.5 ml DNase/RNase-free Microcentrifuge Tubes SS1-1210-0 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. 22