NGS clean-up and size selection - MACHEREY
Contents
1. NGS clean up and size selection User manual NucleoMag NGS Clean up and Size Select May 2014 Rev 01 MACHEREY NAGEL www mn net com NGS clean up and size selection Table of contents 1 Components 1 1 Kit contents 1 2 Equipment and consumables to be supplied by user 4 2 Product description 5 2 1 The basic principle 5 2 2 Kit specifications 5 2 3 Magnetic separation systems 5 2 4 Handling of beads 6 3 Storage conditions and preparation of working solutions 7 4 Safety instructions 7 5 Protocols 8 5 1 Protocol for DNA clean up and single size selection 8 5 2 Protocol for removing adapter dimers 13 5 3 Protocol for DNA double size selection 14 6 Appendix 19 6 1 Troubleshooting 19 6 2 Ordering information 20 6 3 Product use restriction warranty 20 MACHEREY NAGEL 05 2014 Rev 01 3 NGS clean up and size selection 1 Components 1 1 Kit contents NucleoMag NGS Clean up and Size Select 50 100 preps 250 500 preps 2500 5000 preps REF 744970 5 744970 50 744970 500 NucleoMag NGS Bead Suspension 5 mL 50 mL 500 mL User manual 1 1 1 Note The number of preps is calculated according to a sample volume of 50 100 uL and a ratio bead suspension to sample of 1 0 1 2 Equipment and consumables to be supplied by user Reagents 80 ethanol non denatured Elution buffer 10 mM Tris HCl pH 8 or water Consumables Disposable pipette tips Equipment Well calibrated2. The following protocol exemplifys size selection of DNA fragment libraries with a size range of 400 500 bp By altering the volume ratios DNA fragment libraries with other size ranges can be obtained Before starting the preparation Remove the NucleoMag NGS Bead Suspension from the refrigerator Let stand for approxmately 30 min to bring the bead suspension to room temperature 1 Remove unwanted larger DNA fragments Vortex the NucleoMag NGS Bead Suspension well until it appears homogeneous in colour Add 40 uL of well dispersed bead suspension to each well of the separation plate Add 100 uL of DNA sample the volume ratio of binding buffer and bead suspension to sample is 0 4 Adjust the pipette to 140 uL and mix by pipetting up and down 10 times Incubate the separation plate at room temperature for 5 min Separate the magnetic beads against the side of the wells by placing the 96 well plate on the NucleoMag SEP magnetic separator Wait at least 5 min until all the beads have been attracted to the magnets or until the liquid appears clear Transfer the supernatant into the well of a new plate and discard the beads that contain the unwanted large fragments 16 MACHEREY NAGEL 05 2014 Rev 01 NucleoMag NGS Clean up and Size Select Remove unwanted smaller DNA fragments Vortex the NucleoMag NGS Bead Suspension well until it appears homogeneous in colour Add 20 uL of well dispersed bead suspensi
3. Do not disturb the attracted beads while aspirating the supernatant Remove supernatant from the opposite side of the well 10 MACHEREY NAGEL 05 2014 Rev 01 NucleoMag NGS Clean up and Size Select 1 wash with 80 ethanol Leave the 96 well plate on the magnetic separator during the following washing step Add 200 pL 80 ethanol without disturbing the pellet Incubate the separation plate at room temperature for at least 30 s Carefully remove and discard supernatant by pipetting 2 4 wash with 80 ethanol Leave the 96 well plate on the magnetic separator during the following washing step Add 200 uL 80 ethanol without disturbing the pellet Incubate the separation plate at room temperature for at least 30 s Carefully remove and discard supernatant by pipetting Note remove supernatant completely including residual droplets Dry the beads Leave the 96 well plate on the magnetic separator and incubate at room temperature for 5 15 min in order to allow the remaining traces of alcohol to evaporate Note Allow the pellet to dry sufficiently that there are no visible droplets of the supernatant at the bottom of the wells Do not overdry beads Yield may decrease since longer DNA fragments will elute slower MACHEREY NAGEL 05 2014 Rev 01 11 NucleoMag NGS Clean up and Size Select Elute DNA fragment library Remove the 96 well plate from the NucleoMag SEP magnetic separat
4. NGS sequencing applications it is optimal to remove all fragments below 150 200 bp This can be achieved by using a volume ratio bead suspension to sample of 1 0 which is described in the following protocol e g add 100 uL of bead suspension to 100 uL of sample To assure accurate and precise pipetting the sample volume should be 50 uL However volume ratio may be altered to fit the special application of the library construction process Before starting the preparation Remove the NucleoMag NGS Bead Suspension from the refrigerator Let stand for approxmately 30 min to bring the bead suspension to room temperature Bind target DNA to NucleoMag NGS Beads Vortex the NucleoMag NGS Bead Suspension well until it appears homogeneous in colour Add 100 uL of well dispersed bead suspension to each well of the separation plate Add 100 uL of DNA sample the volume ratio of bead suspension to sample is 1 0 Adjust the pipette to 200 uL and mix by pipetting up and down 10 times Incubate the separation plate at room temperature for 5 min Separate the magnetic beads against the side of the wells by placing the 96 well plate on the NucleoMag SEP magnetic separator Wait at least 5 min until all the beads have been attracted to the magnets or until the liquid appears clear Remove and discard supernatant by pipetting The supernatant contains unwanted low molecular weight contaminants and unwanted smaller DNA fragments Note
5. 0 sheets Visit www mn net com for more detailed product information 6 3 Product use restriction warranty NucleoMag NGS Clean up and Size Select kit components are intended developed designed and sold FOR RESEARCH PURPOSES ONLY except however any other function of the product being expressly described in original MACHEREY NAGEL product leaflets MACHEREY NAGEL products are intended for GENERAL LABORATORY USE ONLY MACHEREY NAGEL products are suited for QUALIFIED PERSONNEL ONLY MACHEREY NAGEL products shall in any event only be used wearing adequate PROTECTIVE CLOTHING For detailed information please refer to the respective Material Safety Data Sheet of the product MACHEREY NAGEL products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT MACHEREY NAGEL does not assume any responsibility for damages due to improper application of our products in other fields of application Application on the human body is STRICTLY FORBIDDEN The respective user is liable for any and all damages resulting from such application DNA RNA PROTEIN purification products of MACHEREY NAGEL are suitable for N VITRO USES ONLY ONLY MACHEREY NAGEL products specially labeled as IVD are also suitable for IN VITRO diagnostic use Please pay attention to the package of the product IN VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR IN VITRO DIAGNOSTIC USE ALL O
6. 30 s Remove supernatant carefully 2d wash with Leave the 96 well plate 80 ethanol on NucleoMag SEP 200 uL 80 ethanol Incubate for 30 s Remove supernatant carefully Dry the beads 5 15 min at RT Elute DNA Remove the 96 well plate from NucleoMag SEP 10 50 uL elution buffer Mix by pipetting up and down Incubate for 2 5 min Separate 5 min and transfer DNA into a new 96 well plate MACHEREY NAGEL 05 2014 Rev 01 15 NucleoMag NGS Clean up and Size Select Detailed protocol This protocol can be used to generate DNA fragment libraries with a certain size range or to narrow fragment size distribution The method is called double size selection because both smaller and larger fragments can be removed First an appropriate volume of NucleoMag NGS Bead Suspension is added to the DNA sample This step enables binding of all DNA fragments longer than the desired upper limit of the interval The beads with the unwanted larger DNA fragments are discarded right side selection The supernatant which contains DNA fragments shorter that the upper length cut off is transferred to a new tube to perform the second size selection step left side selection More bead suspension is added to the supernatant so that DNA fragments longer than the lower limit of the interval will be bound After discarding the supernatant DNA fragments within the desired size range are eluted
7. REY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product defects in products or components not manufactured by MACHEREY NAGEL or damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indirect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature ar
8. THER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE 20 MACHEREY NAGEL 05 2014 Rev 01 NGS clean up and size selection No claim or representations is intended for its use to identify any specific organism or for clinical use included but not limited to diagnostic prognostic therapeutic or blood banking It is rather in the responsibility of the user or in any case of resale of the products in the responsibility of the reseller to inspect and assure the use of the DNA RNA protein purification products of MACHEREY NAGEL for a well defined and specific application MACHEREY NAGEL shall only be responsible for the product specifications and the performance range of MN products according to the specifications of in house quality control product documentation and marketing material This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish to get an extra copy There is no warranty for and MACHE
9. and Size Select 5 Dry the beads Leave the 96 well plate on the magnetic separator and incubate at room temperature for 5 15 min in order to allow the remaining traces of alcohol to evaporate Note Allow the pellet to dry sufficiently that there are no visible droplets of the supernatant at the bottom of the wells Do not overdry beads Yield may decrease since longer DNA fragments will elute slower 6 Elute DNA fragment library Remove the 96 well plate from the NucleoMag SEP magnetic separator Add 10 50 uL elution buffer and resuspend the pellet by pipetting up and down 10 times or by shaking e g at 1100 rpm using a thermomixer Incubate the separation plate at room temperature for 2 5 min Note 10 mM Tris HCl pH 8 or water can be used as elution buffer Separate the magnetic beads against the side of the wells by placing the 96 well plate on the NucleoMag SEP magnetic separator Wait at least 5 min until all the beads have been attracted to the magnets or until the liquid appears clear Transfer the supernatant containing the purified DNA fragment library to a new 96 well plate Proceed to the next step of your library preparation process 18 MACHEREY NAGEL 05 2014 Rev 01 NGS clean up and size selection 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Insufficient ratio Use volume ratios outlined in this manual e g 1 0 Insufficient ethanol concentration
10. d target DNA to Mix until suspension is NucleoMag NGS homogeneous Beads 100 pL NucleoMag NGS Beads 100 pL DNA sample Mix by pipetting up and down eke Incubate for 5 min Remove supernatant after 5 min separation 2 1 wash with Leave the 96 well plate 80 ethanol on NucleoMag SEP 200 uL 80 ethanol Incubate for 30 s Remove supernatant carefully 8 MACHEREY NAGEL 05 2014 Rev 01 NucleoMag NGS Clean up and Size Select 2d wash with Leave the 96 well plate 80 ethanol on NucleoMag SEP 200 uL 80 ethanol Incubate for 30 s Remove supernatant carefully Dry the beads 5 15 min at RT Elute DNA Remove the 96 well plate from NucleoMag SEP 10 50 uL elution buffer Mix by pipetting up and down Incubate for 2 5 min Separate 5 min and transfer DNA into a new 96 well plate MACHEREY NAGEL 05 2014 Rev 01 NucleoMag NGS Clean up and Size Select Detailed protocol This protocol can be used to remove contaminants such as nucleotides primers adapters enzymes buffer additives salts and shorter DNA fragments from a sample The method utilizes a single size selection step also called left side selection After adding the appropriate volume of NucleoMag NGS Bead Suspension to the DNA sample beads will bind larger fragments The supernatant contains smaller fragments and contaminants that are discarded For most
11. e MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications MACHEREY NAGEL 05 2014 Rev 01 21 NGS clean up and size selection mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that all applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Last updated 07 2010 Rev 03 Please contact MACHEREY NAGEL GmbH amp Co KG Tel 49 24 21 969 270 tech bio mn net com Trademarks Illumina is a trademark of Illumina Inc and its subsidiaries Eppendorf Thermomixer is a registered trademark of Eppendorf AG Germany NucleoMag is a registered trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands trademarks or registered labels of thei
12. n up and Size Select is designed for rapid manual and automated clean up and size selection of DNA fragments from a variety of reaction mixtures that are used in the library construction process for next generation sequencing such as Fragmentation mixtures End repair mixtures e A tailing mixtures Adapter ligation mixtures PCR amplicifation mixtures The typical sample amount of double stranded DNA fragments is 5 ng to 1 ug By using the tunable size selection method DNA fragment libraries with a size range of 150 bp to 800 bp can be produced To assure accurate and precise pipetting the sample volume should be gt 50 pL The NucleoMag NGS Clean up and Size Select can be processed completely at room temperature 2 3 Magnetic separation systems For use of NucleoMag NGS Clean up and Size Select the use of the magnetic separator NucleoMag SEP see ordering information is recommended Separation is carried out in a 96 well microtiterplate with 300 uL u bottom wells The kit can also be used with other common separators MACHEREY NAGEL 05 2014 Rev 01 5 NGS clean up and size selection 2 4 Handling of beads Liquid handling Precise pipetting of the NucleoMag NGS Bead Suspension and sample is essential for reliable results Variations in volume will affect size selection performance Therefore we recommend to use well calibrated pipettes and new tips after each well single channel or column multichan
13. nel pipette A good technique for pipetting the slightly viscous bead suspension is to pipette very slowly Aspirate slowly and make sure that there are no liquid droplets on the outside of the tip and do not aspirate any air Dispense slowly to ensure that the bead suspension is transferred completely into the wells A homogeneous distribution of the magnetic beads to the individual wells of the separation plate is essential for a high well to well consistency Therefore before distributing the beads make sure that the beads are completely resuspended Shake the storage bottle well or place it on a vortexer shortly Magnetic separation time Attraction of the magnetic beads to the magnetic pins depends on the magnetic strength of the magnetic pins the selected separation plate distance of the separation plate from the magnetic pins and the volume to be processed The individual times for complete attraction of the beads to the magnetic pins should be checked and adjusted on each system It is recommended using the separation plates or tubes specified by the supplier of the magnetic separator Volume ratio NucleoMag NGS paramagnetic beads selectively bind DNA fragments based on the volume ratio of bead suspension and sample In general increasing the volume ratio will favor the adsorption of shorter fragments to the paramagnetic beads This user manual exemplary presents the most commonly used protocols for distinct size range profiles tha
14. on to each well containing supernatants from step 1 the total volume ratio of binding buffer and bead suspension to the original sample is now 0 6 40 uL and 20 uL to 100 uL Adjust the pipette to 160 uL and mix by pipetting up and down 10 times Incubate the separation plate at room temperature for 5 min Separate the magnetic beads against the side of the wells by placing the 96 well plate on the NucleoMag SEP magnetic separator Wait at least 5 min until all the beads have been attracted to the magnets or until the liquid appears clear Remove and discard supernatant by pipetting Note Do not disturb the attracted beads while aspirating the supernatant Remove supernatant from the opposite side of the well 1 wash with 80 ethanol Leave the 96 well plate on the magnetic separator during the following washing step Add 200 uL 80 ethanol without disturbing the pellet Incubate the separation plate at room temperature for at least 30 s Carefully remove and discard supernatant by pipetting 2 4 wash with 80 ethanol Leave the 96 well plate on the magnetic separator during the following washing step Add 200 uL 80 ethanol without disturbing the pellet Incubate the separation plate at room temperature for at least 30 s Carefully remove and discard supernatant by pipetting Note Remove supernatant completely including residual droplets MACHEREY NAGEL 05 2014 Rev 01 17 NucleoMag NGS Clean up
15. or Add 10 50 uL elution buffer and resuspend the pellet by pipetting up and down 10 times or by shaking e g at 1100 rpm using an eppendorf Thermomixer Incubate the separation plate at room temperature for 2 5 min Note 10 mM Tris HCl pH 8 or water can be used as elution buffer Separate the magnetic beads against the side of the wells by placing the 96 well plate on the NucleoMag SEP magnetic separator Wait at least 5 min until all the beads have been attracted to the magnets or until the liquid appears clear Transfer the supernatant containing the purified DNA fragment library to a new 96 well plate Proceed to the next step of your library preparation process 12 MACHEREY NAGEL 05 2014 Rev 01 NucleoMag NGS Clean up and Size Select 5 2 Protocol for removing adapter dimers This protocol can be used to remove adapter dimers after an adapter addition reaction The method utilizes two successive purification steps according to protocol 5 1 In the first step a ratio bead suspension to sample of 1 0 is used to remove DNA precipitating agents from the ligation reaction buffer that interfere with the size selection process The following step eliminates adapter dimers by using the same procedure but with a ratio of 0 8 1 Exchange ligation reaction buffer Perform purification procedure as described in 5 1 with a ratio of 1 0 and elute in 50 pL 2 Remove adapter dimers Perform purification p
16. pipettors Vortex mixer Magnetic separation system e g NucleoMag SEP REF 744900 see section 2 3 Separation plate for magnetic beads separation e g 96 well 0 3 mL microtiterplate Elution Plate U bottom REF 740486 24 e Plate seal e g Self adhering PE Foil REF 740676 4 MACHEREY NAGEL 05 2014 Rev 01 NGS clean up and size selection 2 Product description 2 1 The basic principle The NucleoMag NGS Clean up and Size Select is designed for rapid clean up and size selection of DNA fragments in the library construction process for next generation sequencing NGS The NucleoMag NGS Bead Suspension contains paramagnetic beads that are suspended in a special binding buffer Paramagnetic beads selectively bind DNA fragments based on the volume ratio of bead suspension and sample After magnetic separation and removal of supernatant the beads are washed with ethanol A short drying step is necessary to remove ethanol from previous washing steps Finally highly purified DNA fragments are eluted with low salt elution buffer or water that can be used directly for downstream applications The purified DNA fragment library is free of any contaminants such as nucleotides primers adapters adapter dimers enzymes buffer additives and salts The NucleoMag NGS Clean up and Size Select kit can be used either manually or automated on standard liquid handling instruments 2 2 Kit specifications NucleoMag NGS Clea
17. r respective owner also if they are not special denotation To mention products and brands is only a kind of information i e it does not offend against trademarks and brands and can not be seen as a kind of recommendation or assessment Regarding these products or services we cannot grant any guarantees regarding selection efficiency or operation 22 MACHEREY NAGEL 05 2014 Rev 01
18. rocedure as described in 5 1 but with a ratio of 0 8 to 50 uL of eluate from step 1 add 40 uL of NucleoMag NGS Bead Suspension Elute in 30 uL Proceed to the next step of your library construction process MACHEREY NAGEL 05 2014 Rev 01 13 NucleoMag NGS Clean up and Size Select 5 3 Protocol for DNA double size selection Protocol at a glance For additional equipment and hardware requirements refer to section 1 2 and 2 3 respectively For detailed information on each step see page 16 Before starting the preparation Remove the NucleoMag NGS Bead Suspension from the refrigerator Let stand for approxmately 30 min to bring the bead suspension to room temperature 1 Remove unwanted Mix until suspension is larger DNA fragments homogeneous 40 pL NucleoMag NGS Beads 100 pL DNA sample Mix by pipetting up and down Incubate for 5 min Remove and safe supernatant after 5 min separation Transfer supernatant into a new 96 well plate Discard beads 2 Remove unwanted 20 uL NucleoMag NGS Beads smaller DNA to supernatant of step 1 fragments Mix by pipetting up and down Incubate for 5 min Remove and discard supernatant after 5 min separation 14 MACHEREY NAGEL 05 2014 Rev 01 NucleoMag NGS Clean up and Size Select 1 wash with Leave the 96 well plate 80 ethanol on NucleoMag SEP 200 uL 80 ethanol Incubate for
19. t are optimal for NGS applications using llumina sequencing systems By altering the volume ratio DNA fragment libraries with a size range of 150 bp to 800 bp for any sequencing platform can be produced The NucleoMag NGS Bead Suspension is similar to other well known producs in the market Therefore you can use the same volume ratios that are recommended in your NGS library Kit preparation protocol 6 MACHEREY NAGEL 05 2014 Rev 01 NGS clean up and size selection 3 Storage conditions and preparation of working solutions The NucleoMag NGS Clean up and Size Select kit is shipped at ambient temperature The bead suspension should be stored at 4 8 C upon arrival and is stable for up to one year under proper storage conditions The NucleoMag NGS Bead Suspension is delivered ready to use 4 Safety instructions The NucleoMag NGS Clean up and Size Select kit does not contain hazardous contents MACHEREY NAGEL 05 2014 Rev 01 7 NucleoMag NGS Clean up and Size Select 5 Protocols 5 1 Protocol for DNA clean up and single size selection Protocol at a glance For additional equipment and hardware requirements refer to section 1 2 and 2 3 respectively For detailed information on each step see page 10 Before starting the preparation Remove the NucleoMag NGS Bead Suspension from the refrigerator Let stand for approxmately 30 min to bring the bead suspension to room temperature 1 Bin
20. used for washing step Use freshly prepared 80 ethanol Over time ethanol becomes more dilute through evaporation and absorption of atmospheric water As a consequence parts of the DNA pellet goes into solution and DNA fragments are washed away Poor DNA yield Elution buffer volume insufficient Bead pellet must be covered completely with elution buffer Incubation time for elution insufficient Incubate beads in elution buffer for 5 min for optimal yields Beads overdried Do not dry beads longer than 15 min at room temperature Overdrying of beads may result in lower elution efficiencies Suboptimal Carry over of ethanol from washing step performance Be sure to remove all of the ethanol after the final washing of DNA in step Dry beads 5 10 min at room temperature downstream applications Carry over of beads Time for magnetic separation too short Increase separation time to allow the beads to be attracted to the magnetic pins completely Aspiration speed too high elution step High aspiration speeds during the elution step may cause bead carry over Reduce aspiration speed for elution step MACHEREY NAGEL 05 2014 Rev 01 19 NGS clean up and size selection 6 2 Ordering information Product REF Pack of NucleoMag NGS Clean up and 744970 5 1 x 96 preps Size Select 744970 50 4 x 96 preps 744970 500 24 x 96 preps NucleoMag SEP 744900 1 Elution Plate U bottom 740486 24 24 Self adhering PE Foil 740676 5
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