PCR clean-up Gel extraction User manual
Contents
1. H 300 pL NT1 4 uL silica matrix ug DNA 50 C 5 10 min 10 000 x g c 30s 10 000 x g 30s li 500 pL NT2 c 12 MACHEREY NAGEL 03 2014 Rev 06 NucleoTrap Add 500 uL Buffer NT3 to the pelleted silica matrix and vortex briefly Centrifuge for 30 s at 10 000 x g and remove the supernatant completely Add 500 uL Buffer NT3 to the pelleted silica matrix and vortex briefly Centrifuge for 30 s at 10 000 x g Remove the supernatant and centrifuge the pellet again briefly Remove residual Buffer NT3 completely Dry silica matrix Dry the pelleted silica matrix at room temperature or at 37 C for 10 15 min It is not recommended to dry the sample by vacuum since over dried pellets lead to lower recoveries Residual ethanol from Buffer NT3 would inhibit subsequent reactions and has to be removed in this step Elute DNA Add 25 50 uL Buffer NE to the silica matrix Resuspend the pellet by vortexing Incubate the mixture at room temperature for 10 15 min Vortexing the mixture 2 3 times during incubation is recommended Centrifuge the sample at 10 000 x g for 30 s and transfer the DNA containing supernatant to a clean tube not provided Repeating this step will increase the yield by approximately 10 Yield of larger fragments gt 5 20 kbp can be increased by performing the incubation at 55 C ec J 500 pL NT3 10 000 x g 30s 500 pL NT3 10 000 x g 30s RT or2. PCR clean up Gel extraction User manual NucleoTraP CR NucleoTraP March 2014 Rev 06 MACHEREY NAGEL www mn net com PCR clean up gel extraction Protocol at a glance Rev 06 NucleoTraP CR NucleoTrap PCR clean up Gel extraction 1 NucleoTrap Excise DNA fragment Solubilize gel slice NucleoTraP CR Adjust binding conditions 4 vol NT2 300 uL NT1 1 vol sample 100 mg gel b 10 000 x g 30s 2 Bind DNA 10 uL silica matrix 4 uL silica matrix 100 uL sample ug DNA RT 50 C 10 min 5 10 min 10 000 x g 10 000 x g 30s 30s 3 Wash silica matrix EH 400 pL NT2 EH 500 uL NT2 EW cuv H ES soo L NT3 EW 400 uL NT EW uw 10 000xg 30s 10 000xg 30s 10 000xg 30s 3 10 000xg 30s 10 000xg 30s 4 Dry silica matrix RT or 37 C RT or 37 C 10 15 min 10 15 min 5 Elute DNA 25 50 uL NE 25 50 uL NE RT RT 10 15 min 10 15 min 10 000 x g 10 000 x g 30s e 80s MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 52355 D ren Germany Tel 49 24 21 969 270 Fax 49 24 21 969 199 tech bio mn net com www mn net com PCR clean up gel extraction Table of contents 1 Components 4 1 1 Kit contents 4 1 2 Consumables and equipment to be supplied by the user 5 1 3 About this user manual 5 2 Product description 6 2 1 The basic principle 6 2 2 Kit specifications 6 2 3 Elution procedures 7 3 Storage conditions and preparatio
3. 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 IN 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 OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE 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 wel
4. matrix the NucleoTraP CR matrix will not bind DNA fragments 100 bp due to a larger pore size of the silica matrix Standard as well as low melting agarose gels can be used The prepared DNA fragments can be used directly in applications like automated fluorescent DNA sequencing PCR or any kind of enzymatic manipulation Table 1 Kit specifications at a glance Parameter NucleoTraP CR NucleoTrap Technology Silica matrix Silica matrix Format Silica bead suspension Silica bead suspension Sample material lt 400 uL PCR reaction lt 200 mg agarose gel mixture DNA fragments from agarose gels Desalting removal of enzymes nucleotides and or labeling reagents like biotin or radioactive ATP etc Direct purification of amplified DNA not recommended possible optimal 6 MACHEREY NAGEL 03 2014 Rev 06 PCR clean up gel extraction Table 1 Kit specifications at a glance Parameter NucleoTraP CR NucleoTrap Fragment size 100 bp approx 50kbp 20 bp approx 50 kbp Typical recovery 70 80 96 50 90 A260 A280 1 7 1 9 1 7 1 9 Elution volume 20 50 uL 20 50 uL Preparation time 45 min 6 preps 60 min 6 preps Binding capacity 6 ug 10 uL matrix 6 ug 10 uL matrix 2 3 Elution procedures For the elution of DNA one of the following solutions can be used Buffer NE supplied TE buffer pH 8 5 distilled water pH 8 5 If water is used the pH should be checked and adjuste
5. 37 C 10 15 min 20 50 uL NE 10 000 x g 30s MACHEREY NAGEL 03 2014 Rev 06 13 NucleoTraP CR NucleoTrap 7 Protocol for concentration desalting removal of enzymes etc Before starting the preparation Check if Wash Buffer NT3 was prepared according to section 3 1 Adjust DNA binding conditions 4 vol NT2 Add 4 volumes Buffer NT2 to 1 volume of DNA per containing sample e g 400 uL Buffer NT2 and 100 uL 1 vol sample reaction mixture 2 Bind DNA Vortex the NucleoTraP CR NucleoTrap Suspension 4 pL silica thoroughly until a homogeneous mixture results For matrix each ug of DNA add 4 pL of silica matrix but at least g DNA 10 uL RT Incubate the mixture for 10 min at room temperature and 10 min vortex briefly every 2 3 min 10 000 x g e s Centrifuge for 30 s at 10 000 x g and discard supernatant 30s Important note Be aware of the NucleoTrap Suspension binding fragments down to 20 bp see Table 2 section 2 3 Continue with section 5 step 3 14 MACHEREY NAGEL 03 2014 Rev 06 PCR clean up gel extraction 8 Appendix 8 1 Troubleshooting Problem Possible cause and suggestions High concentration of agarose Use doubled volumes of Buffer NT1 for highly concentrated agarose gels c Wrong buffer ncomplete lysis Ri Buffer NT2 cannot be used for gel dissolution agarose slices Time and temperature e Check incubation temperature Depend
6. E 10 000 x g 1 min MACHEREY NAGEL 03 2014 Rev 06 11 NucleoTraP 6 NucleoTrap protocol DNA extraction from agarose gels Before starting the preparation Check if Wash Buffer NT3 was prepared according to section 3 Set heating block to 50 C Excise DNA fragment Solubilize gel slice Take a clean scalpel to excise the DNA fragment from agarose gel Excise gel slice containing the fragment carefully to minimize the gel volume Determine the weight of the gel slice and transfer it to a clean tube not provided For each 100 mg agarose gel add 300 pL NT1 For gels containing gt 2 agarose double the volume of Buffer NT1 Note If the weight of the gel slice is 100 mg the volume of Buffer NT1 must be increased proportionally Example a 150 mg gel slice lt 2 agarose needs 450 uL Buffer NT1 Bind DNA Vortex the NucleoTrap Suspension thoroughly until a homogeneous mixture results For each pg of DNA add 4 uL of the NucleoTrap Suspension but at least 10 uL Incubate sample at 50 C until the gel slice is dissolved 5 10 min Vortex the sample briefly every 2 3 min until the gel slice is dissolved completely Centrifuge for 30 s at 10 000 x g and discard supernatant Wash silica matrix Add 500 uL Buffer NT2 to the pelleted silica matrix and vortex briefly for resuspension of the pellet Centrifuge for 30s at 10 000x g and remove the supernatant completely
7. HE 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 are 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 y
8. HEREY NAGEL 03 2014 Rev 06 PCR clean up gel extraction 4 Safety instructions The following components of the NucleoTraP CR NucleoTrap kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section GHS classification Only harmful features do not need be labeled with H and P phrases until 125 mL or 125 g Mindergef hrliche Eigenschaften m ssen bis 125 mL oder 125 g nicht mit H und P S tzen gekennzeichnet werden Component Hazard contents GHS symbol Hazard Precaution phrases phrases Inhalt Gefahrstof GHS Symbol H S tze P S tze NT1 NT2 Sodium perchlorate 40 60 D Danger 302 210 220 Natrium perchlorat 40 60 Gefahr 301 312 330 Hazard phrases H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken Precaution phrases P 210 Keep away from heat hot surfaces sparks open flames and other ignition sources No smoking VVon Hitze hei en Oberfl chen Funken offenen Flammen sowie anderen Z ndquellenarten fernhalten Nicht rauchen P 220 Keep Store away from clothing combustible materials Von Kleidung brennbaren Materialien fernhalten entfernt aufbewahren P 301 312 IF SWALLOWED Call a POISON CENTER or doctor physician if you feel unwell BEI VERSCHLUCKEN Bei Unwohlsein GIFTINFORMATIONSZENTRUM oder Arzt anrufen P 330 Rinse mouth Mund aussp len For further information please see Material Safety Data Sheets www mn
9. d to pH 8 8 5 since deionized water usually exhibits a pH below 7 Furthermore absorption of CO leads to a decrease in pH of unbuffered solutions Note EDTA in TE buffer may cause problems in subsequent reactions See Table 2 for correlation between fragment size and typical recoveries for purification of 1 5 ug of PCR fragments for gel extraction recoveries are approximately 10 lower Table 2 DNA recovery with NucleoTraP CR NucleoTrap Fragment length NucleoTraP CR NucleoTrap 20 bp 0 50 40 bp 0 68 120 bp 68 78 200 bp 76 85 520 bp 80 87 2 5 kbp 81 88 5 3 kbp 80 86 8 7 kbp 76 80 19 4 kbp 74 74 MACHEREY NAGEL 03 2014 Rev 06 7 PCR clean up gel extraction 3 Storage conditions and preparation of working solutions Attention Buffers NT1 and NT2 contain chaotropic salts Wear gloves and goggles The NucleoTraP CR NucleoTrap kits should be stored at room temperature and are stable for up to one year Before starting any NucleoTraP CR NucleoTrap protocol prepare the following Wash Buffer NT3 Add the indicated volume of 96 100 ethanol to Wash Buffer NT3 Concentrate NucleoTraP CR 10 preps 100 preps REF 740587 10 740587 Wash Buffer NT3 6 mL 25 mL Concentrate Add 24 mL ethanol Add 100 mL ethanol NucleoTrap 10 preps 100 preps REF 740584 10 740584 Wash Buffer NT3 6 mL 25 mL Concentrate Add 24 mL ethanol Add 100 mL ethanol MAC
10. ead the detailed protocol sections of this user manual if using the NucleoTraP CR NucleoTrap kits for the first time However experienced users may refer to the Protocol at a glance The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure All technical literature is available on the internet at www mn net com Please contact Technical Service regarding information about changes of the current user manual compared to previous revisions MACHEREY NAGEL 03 2014 Rev 06 5 PCR clean up gel extraction 2 Product description 2 1 The basic principle With the NucleoTraP CR Trap method DNA binds in the presence of chaotropic salts Buffer NT1 and Buffer NT2 to specially activated silica particles matrix Buffer NT1 contains additional components in order to dissolve agarose gel slices Afterwards the NucleoTraP CR Trap matrix is added to the binding mixtures Contaminations like salts and soluble macromolecular components are removed by a simple washing step with ethanolic Wash Buffer NT3 Pure DNA is finally eluted under low ionic strength conditions with slightly alkaline Elution Buffer NE 5 mM Tris Cl pH 8 5 2 2 Kit specifications The NucleoTraP CR kit is designed for direct purification of PCR products The NucleoTrap kit is designed for the purification of DNA from TAE TBE agarose gels Incontrast to the NucleoTrap
11. ica matrix RT 10 min 10 000 x g 30s e 400 uL NT2 10 000 x g 30s 10 MACHEREY NAGEL 03 2014 Rev 06 NucleoTraP CR Add 400 uL Buffer NT3 to the pelleted silica matrix and vortex briefly Centrifuge for 30 s at 10 000 x g and remove the supernatant completely Add 400 uL Buffer NT3 to the pelleted silica matrix and vortex briefly Centrifuge for 30 s at 10 000 x g Remove the supernatant and centrifuge the pellet again briefly Remove residual Buffer NT3 completely Dry silica matrix Dry the pelleted silica matrix at room temperature or at 37 C for 10 15 min It is not recommended to dry the sample by vacuum since over dried pellets lead to lower recoveries Residual ethanol from Buffer NT3 would inhibit subsequent reactions and has to be removed in this step Elute DNA Add 25 50 uL Buffer NE to the silica matrix Resuspend the pellet by vortexing Incubate the mixture at room temperature for 10 15 min Vortexing the mixture 2 3 times during incubation is recommended Centrifuge the sample at 10 000 x g for 30 s and transfer the DNA containing supernatant to a clean tube not provided Repeating this step will increase the yield by approximately 10 Yield of larger fragments gt 5 20 kbp can be increased by performing the incubation at 55 C Il 400 uL NT3 10 000 x g 30s 400 uL NT3 10 000 x g 30s RT or 37 C 10 15 min 25 50 uL N
12. ing on the weight of gel slice incubation section 6 step 2 can be prolonged up to 20 min Vortex every 2 min and check integrity of the gel slice Heavy weight gel slices may be quenched or crushed before addition of Buffer NT1 Reagents not applied properly Add indicated volume of 96 100 ethanol to Wash Buffer NT3 Concentrate and mix well before use No DN Insufficient drying of the Nucleo TraP CR NucleoTrap silica matrix yield Ethanolic Wash Buffer NT3 has to be removed quantitatively before elution Prolong the drying time up to 30 min Ethanolic contaminations are also indicated by gel loading problems samples float out of gel slots Isolation of large DNA fragments Add room temperature Elution Buffer NE and incubate at 55 C for 10 15 min MACHEREY NAGEL 03 2014 Rev 06 15 PCR clean up gel extraction Problem Possible cause and suggestions Carry over of ethanol ethanolic Buffer NT3 Make sure to dry the silica matrix in order to achieve complete removal of ethanolic Buffer NT3 after the washing step Ethanolic contaminations are also indicated by gel loading problems samples float out of gel slots Buffers other than Buffer NE for example TE buffer Tris EDTA were usedfor elution of DNA Note EDTA may inhibit sequencing Suboptimal reactions In this case it is recommended to re purify DNA and performance elute in Buffer NE or water of DNA in sequencin sa lon s g Not enough DNA used fo
13. l 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 MACHEREY 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 18 MACHEREY NAGEL 03 2014 Rev 06 PCR clean up gel extraction 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 T
14. n of working solutions 8 4 Safety instructions 9 5 NucleoTraP CR protocol direct purification of PCR products 10 6 NucleoTrap protocol DNA extraction from agarose gels 12 7 Protocol for concentration desalting removal of enzymes etc 14 8 Appendix 15 8 1 Troubleshooting 15 8 2 Ordering information 17 8 3 References 17 8 4 Product use restriction warranty 18 MACHEREY NAGEL 03 2014 Rev 06 3 PCR clean up gel extraction 1 Components 1 1 Kit contents NucleoTraP CR 10 preps 100 preps REF 740587 10 740587 NucleoTraP CR Suspension 100 uL 1000 uL Buffer NT2 10 mL 100 mL iConcontate om A Elution Buffer NE 13 mL 13 mL User manual 1 1 10 preps 100 preps REF 740584 10 740584 NucleoTrap Suspension 100 uL 1000 uL Buffer NT1 6mL 2x30mL Buffer NT2 10 mL 100 mL er sm in Elution Buffer NE 13 mL 13 mL User manual 1 1 For preparation of working solutions and storage conditions see section 3 Composition of Elution Buffer NE 5 mM Tris HCl pH 8 5 gt MACHEREY NAGEL 03 2014 Rev 06 PCR clean up gel extraction 1 2 Consumables and equipment to be supplied by the user Consumables 96 100 ethanol 1 5 mL microcentrifuge tubes Equipment Centrifuge for microcentrifuge tubes Manual pipettors and disposable tips Vortex mixer Heating block Personal protection equipment lab coat gloves goggles 1 3 About this user manual It is strongly recommended to r
15. net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com MACHEREY NAGEL 03 2014 Rev 06 9 NucleoTraP CR 5 NucleoTraP CR protocol direct purification of PCR products Before starting the preparation Check if Wash Buffer NT3 was prepared according to section 3 Adjust DNA binding conditions Add 4 volumes of Buffer NT2 to 1 volume of sample e g 400 uL Buffer NT2 and 100 uL PCR reaction mixture For sample volumes lt 100 uL adjust the volume of the reaction mix to 100 uL using TE buffer pH 7 5 Note If the volume of the PCR reaction mixture is gt 100 uL the volumes of Buffer NT2 and NucleoTraP CR Suspension must be increased proportionally Example a volume of 150 uL reaction mixture needs 600 uL of Buffer NT2 and 15 uL NucleoTraP CR Suspension to adjust proper binding conditions 2 Bind DNA Vortex the NucleoTraP CR Suspension thoroughly until a homogeneous mixture results Add 10 pL of NucleoTraP CR Suspension to each 100 pL of reaction mixture Incubate the mixture for 10 min at room temperature and vortex briefly every 2 3 min Centrifuge the sample at 10 000 x g for 30 s and discard the supernatant 3 Wash silica matrix Add 400 uL Buffer NT2 to the pelleted silica matrix and vortex briefly for resuspension of the pellet Centrifuge for 30s at 10 000x g and remove the supernatant completely 4 vol NT2 per 1 vol sample 10 pL sil
16. our local distributor for general scientific information Applications 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 9 mn net com Patents Trademarks PCR is patented by Roche Diagnostics NucleoTrap is a trademark of MACHEREY NAGEL GmbH amp Co KG All used names and denotations can be brands trademarks or registered labels of their 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 can not grant any guarantees regarding selection efficiency or operation MACHEREY NAGEL 03 2014 Rev 06 19
17. r sequencing reaction Quantitate DNA by agarose gel electrophoresis before setting up sequencing reactions NucleoTraP CR or NucleoTrap particles were not removed quantitatively Centrifuge the eluate again and transfer the supernatant to a new tube 16 MACHEREY NAGEL 03 2014 Rev 06 PCR clean up gel extraction 8 2 Ordering information Product REF Pack of Nuclear on 74087 a NucleoTrap 740884 S arene NucleoTraP CR Suspension 740564 100 preps NucleoTrap Suspension 740569 100 preps Buffer NT1 740596 100 2x50 mL Buffer NT2 740597 2x50 mL Buffer NT3 Concentrate 740598 20 mL for 100 mL Buffer NT3 Collection Tubes 2 mL 740600 1000 Visit www mn net com for more detailed product information 8 3 References Vogelstein B and D Gillespie 1979 Preparative and analytical purification of DNA from agarose Proc Natl Acad Sci USA 76 615 619 MACHEREY NAGEL 03 2014 Rev 06 17 PCR clean up gel extraction 8 4 Product use restriction warranty NucleoTraP CR NucleoTrap 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
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