PCR clean-up Gel extraction User Manual
Contents
1. PCR clean up Gel extraction User Manual NucleoSpin Extract II Plesman laan 1d 2333 BZ Leiden The Netherlands z E Q B E T 31 0 71 568 10 00 T Belgium 0800 71640 sharing knowledge c 31 0 71 568 10 10 info bioke com www bioke com January 2011 Rev 11 MACHEREY NAGEL MN PCR clean up Gel extraction Protocol at a glance Rev 11 NucleoSpin Extract II PCR clean up Gel extraction 1 PCR clean up Adjust binding condition E Gel extraction Excise DNA fragment 200 uL NT 200 uL NT Solubilize gel slice 100 uL PCR 100 mg gel 50 C 5 10 min 2 Bind DNA 11 000 x g 1 min 3 Wash silica membrane 700 uL NT3 11 000 x g 1 min 4 Dry silica membrane 11 000x g 2 min 5 Elute DNA 15 50 uL NE RT 1 min 11 000 x g 1 min 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 1 1 Kit contents 1 2 Reagents consumables and equipment to be supplied by user 1 8 About this User Manual 2 Product description 2 1 The basic principle 2 2 Kit specifications 2 3 Removal of small DNA fragments and primer dimers 2 4 Elution procedures 3 Storage conditions and preparation of working solutions 4 Safety instructions risk and safety phrases 5 Protoc2. 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 N VITRO diagnostic use Please pay attention to the package of the product N VITRO diagnostic products are expressly marked as IVD on the packaging IF THERE IS NO IVD SIGN THE PRODUCT SHALL NOT BE SUITABLE FOR N 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 th
3. User Manual if the NucleoSpin Extract II kit is used for the first time Experienced users however may refer to the Protocol at a glance instead 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 MACHEREY NAGEL 01 2011 Rev 11 5 PCR clean up Gel extraction 2 2 1 Product description The basic principle With the NucleoSpin Extract Il method DNA binds to a silica membrane in the presence of chaotropic salt added by Binding Buffer NT The binding mixture is loaded directly onto NucleoSpin Extract Il Columns 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 HCl pH 8 5 2 2 Kit specifications The NucleoSpin Extract Il kit is designed for the direct purification of PCR products and for the purification of DNA from TAE TBE agarose gels two applications in one kit The NucleoSpin Extract Il buffer formulation ensures complete removal of primers from PCR reactions while small DNA fragments are still bound and purified with high recovery With NucleoSpin Extract Il even DNA fragments from DCH reaction buffers rich in various detergents can be purifie
4. mL 2x6mL 40 mL Concentrate Add 24 mL ethanol Add 24 mL ethanol Add 160 mL ethanol to each bottle MACHEREY NAGEL 01 2011 Rev 11 11 PCR clean up Gel extraction 4 Safety instructions risk and safety phrases The following components of the NucleoSpin Extract Il kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section Component Hazard Hazard Risk Safety contents symbol phrases phrases NT Guanidinium x Xn Harmful by inhala R 20 21 22 8 18 thiocyanate tion in contact with skin and if swal lowed Risk phrases R 20 21 22 Harmful by inhalation in contact with skin and if swallowed Safety phrases S 13 Keep away from food drink and animal feedstuffs Hazard labeling not necessary if quantity per bottle below 125g or mL certificate of exemption according to 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 8 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet 12 MACHEREY NAGEL 01 2011 Rev 11 NucleoSpin Extract II 5b Protocol for PCR clean up The following protocol is suitable for PCR clean up as well as concentration and removal of salts enzymes etc from samples without SDS Before starting the preparation Check if Wash Buffer NT3 was prepared according to section 3 1 Adjust DNA binding condition For sample volumes 100 uL adjust the volume of the reaction mix to 100 u
5. most up to date information on MACHEREY NAGEL products You may also contact your 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 e mail tech bio mn net com MACHEREY NAGEL 01 2011 Rev 11 23 PCR clean up Gel extraction Trademarks NanoDrop is a registered trademark of NanoDrop Technologies Inc NucleoSpin is a registered 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 24 MACHEREY NAGEL 01 2011 Rev 11
6. of gel slots Remove the spin cup carefully from the centrifuge and collection tube and avoid contacting the spin cup with the flow through Carry over of chaotropic salts Modify washing and or drying step in case of sensitive downstream applications to remove last traces of Buffer NT Note The volume of Buffer NT3 included in the kit might not be sufficient when performing these modifications Buffer NT3 can be ordered separately see ordering information 1 Add 250 uL Buffer NT3 to the NucleoSpin Extract Il Column at the drying step section 5 or 6 step 4 2 Perform a second washing step with 700 uL Buffer NTS section 5 or 6 step 3 and add 250 uL Buffer NT3 at the drying Step section 5 or 6 step 4 Elution of DNA with buffers other than Buffer NE for example TE buffer Tris EDTA EDTA might inhibit sequencing reactions In this case it is recommended to re purify DNA and elute in Buffer NE or water Not enough DNA used for sequencing reaction Quantify DNA by agarose gel electrophoresis before setting up sequencing reactions 20 MACHEREY NAGEL 01 2011 Rev 11 PCR clean up Gel extraction Problem Possible cause and suggestions Suboptimal DNA was damaged by UV light performance Reduce UV exposure time to a minimum when excising a DNA of DNA in fragment from an agarose gel sequencing restriction or ligation reactions continued Suboptimal Carry over of traces of silica particle
7. 5 distilled water pH 8 5 or comparable low salt buffer important pH gt 7 Note EDTA in TE buffer may cause problems in subsequent reactions and the pH of distilled water should be checked before use to avoid lower recovery See Table 2 for the correlation between dispensed elution buffer volumes and typical recoveries for the purification of 1 5 ug of PCR fragments for gel extraction recovery is approx 10 lower With an elution volume of 15 uL of Buffer NE a typical recovery of 70 95 is usually obtained for DNA fragments between 50 10 000 bp resulting in highly concentrated eluates see Table 2 Figure 2 If larger amounts 5 15 ug of DNA have to be purified e g from PCR reactions gt 100 uL or gel slices gt 200 mg elution with at least 50 uL of Buffer NE is recommended Primers are not bound Yields of larger fragments gt 5 10 kbp can be increased by using preheated elution buffer 70 C For elution add preheated Elution Buffer NE and incubate for 1 2 min before collecting eluate by centrifugation For fragments gt 10 kbp the use of our NucleoTraP CR kit is recommended Table 2 DNA recovery with NucleoSpin Extract Il Fragment length Elution volume Recovery 15 uL 8596 25 uL 90 96 63 bp 50 pL 95 100 uL 9596 15 uL 85 96 25 uL 9596 400 bp 50 pL 100 100 uL 10096 15 uL 85 96 25 uL 90 96 700 Bp 50 uL 9595 100 uL 9596 15 uL 8596 25 uL 8596 1500 bp 50 pL 90 100 uL 9596 P
8. CR is patented by Roche Diagnostics MACHEREY NAGEL 01 2011 Rev 11 9 PCR clean up Gel extraction Reference ladder Elution volume pL Did ml Ole 015 br Ab ar re een ee Feet Fee ren Seen E en Soss Jl m Fee Recovery 75 ae 300 Figure 2 DNA recovery with different elution volumes A PCR sample with a fragment size of 782 bp was purified from a 1 TAE agarose gel according to the standard protocol of NucleoSpin Extract Il using different elution volumes as shown All elution volumes were adjusted to 25 uL plus 4 5 uL loading dye For analysis the mixture was loaded on a 1 TAE agarose gel The recovery was estimated by comparison with a fragment ladder 10 MACHEREY NAGEL 01 2011 Rev 11 PCR clean up Gel extraction 3 Storage conditions and preparation of working solutions Attention Buffer NT contains chaotropic salt Wear gloves and goggles Storage conditions The NucleoSpin Extract Il kit should be stored at room temperature and is stable for at last one year Before starting any NucleoSpin Extract II protocol prepare the following Wash Buffer NT3 Add the indicated volume of ethanol 96 100 to Buffer NT3 Concentrate Mark the label of the bottle to indicate that ethanol was added Wash Buffer NT3 is stable at room temperature 18 25 C for at least one year NucleoSpin Extract Il 10 preps 50 preps 250 preps REF 740609 10 740609 50 740609 250 Wash Buffer NT3 6
9. ECTLY 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 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
10. L using water 2 vol NT Mix 1 volume of sample with 2 volumes of Buffer NT per e g mix 100 uL PCR reaction and 200 uL Buffer NT 1 vol sample Note For removal of DNA fragments gt 65 bp dilutions of Buffer NT can be used instead of 100 Buffer NT Please refer to section 2 3 2 Bind DNA Place a NucleoSpin Extract Il Column into a Collection Load sample Tube 2 mL and load the sample Centrifuge for 1 min at 11 000 x g Discard flow through 11 000 xg and place the column back into the collection tube 1 min 3 Wash silica membrane Add 700 uL Buffer NT3 to the NucleoSpin Extract II Column Centrifuge for 1 min at 11 000 x g Discard flow through and place the column back into the collection 700 uL NT3 tube Note Carry over of chaotropic salt may result in low A A values To prevent problems in very sensitive downstream de 11 000 xg applications or if the entire eluate has to be used follow the 1 min instructions given in section 8 1 Suboptimal performance of DNA in sequencing restriction or ligation reactions Carry over of chaotropic salts MACHEREY NAGEL 01 2011 Rev 11 13 NucleoSpin Extract II Dry silica membrane Centrifuge for 2 min at 11 000 x g to remove Buffer NT3 completely Make sure the spin column does not come in contact with the flow through while removing it from the centrifuge and the collection tube Note Residual ethanol from Buffer NT3 might inhib
11. cleoSpin Extract II Column into a new RT 1 5 mL microcentrifuge tube not provided Add 15 1 min 50 uL Buffer NE and incubate at room temperature 18 25 C for 1 min Centrifuge for 1 min at 11 000 x g Note Yield of larger fragments gt 5 10 kbp can be increased ed 11 000 x g by using prewarmed elution buffer 70 C 1 min MACHEREY NAGEL 01 2011 Rev 11 NucleoSpin Extract II 7 Support protocols 7 1 Purification of samples containing SDS Buffer NTB The NucleoSpin Extract Buffer NT is compatible with most commonly used detergents except sodium dodecyl sulfate SDS For purification of DNA from samples without SDS the standard protocol for PCR clean up can be used see section 5 For purification of DNA from SDS containing buffers for example in applications like Chromatin Immunoprecipitation ChIP the SDS compatible Binding Buffer NTB can be used Note Buffer NTB has to be ordered separately 150 mL Buffer NTB REF 740595 150 see ordering information Before starting the preparation Check if Wash Buffer NT3 was prepared according to section 3 1 Adjust DNA binding condition Mix 1 volume of sample with 5 volumes of Buffer NTB 5 vol NTB e g 100 pL reaction mix with 500 uL Buffer NTB per 1 vol sample Note If SDS starts to precipitate add 1 volume of isopropanol or warm sample to 20 30 C 2 Bind DNA Continue with step 2 of the protocol for direct purificatio
12. d with high recovery The adsorption of DNA to the NucleoSpin Extract ll membrane is pH dependent Optimal recovery is achieved by using TAE standard gels or reaction mixtures with pH 6 8 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 reaction PCR is patented by Roche Diagnostics 6 MACHEREY NAGEL 01 2011 Rev 11 PCR clean up Gel extraction Table 1 Kit specifications at a glance Parameter NucleoSpin Extract Il Elution volume 15 50 uL Binding capacity 15 ug Time prep 10 min for 6 preps Direct purification of amplified DNA see section 5 Concentration removal of salts enzymes nucleotides and or labeling reagents like see section 5 and 7 1 biotin or radioactive ATP etc DNA fragments from agarose gels see section 6 Purification of reaction mixtures without SDS Purification of reaction mixtures containing SDS see section 5 see section 7 1 Purification of single stranded DNA see section 7 2 Removal of small DNA fragments and primer dimers see section 2 3 2 3 Removal of small DNA fragments and primer dimers NucleoSpin Extract Il is designed to remove even traces of unused primers and at the same time to purify PCR products down to 65 bp However in some cases it is necessary to exclude these small fragments e g prime
13. e 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 22 MACHEREY NAGEL 01 2011 Rev 11 PCR clean up Gel extraction 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 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 DIR
14. it S 11 000 x g enzymatic reactions Total removal of ethanol can be 2 min achieved by incubating the columns for 2 5 min at 70 C prior to elution Elute DNA 15 50 uL NE Place the NucleoSpin Extract II Column into a new 1 5 mL microcentrifuge tube not provided Add 15 50 pL Buffer NE and incubate at room temperature 18 25 C for 1 min Centrifuge for 1 min at 11 000 x g RT 1 min Note Yield of larger fragments gt 5 10 kbp can be increased e 11 000 x g by using prewarmed elution buffer 70 C 1 min 14 MACHEREY NAGEL 01 2011 Rev 11 NucleoSpin Extract II 6 Protocol for DNA extraction from agarose gels Before starting the preparation Check if Wash Buffer NT3 was prepared according to section 3 1 Excise DNA fragment Solubilize gel slice Take a clean scalpel to excise the DNA fragment from an agarose gel Excise gel slice containing the fragment carefully to minimize the gel volume Note Minimize UV exposure time to avoid damaging the DNA Determine the weight of the gel slice and transfer it to a clean tube For each 100 mg of agarose gel add 200 uL Buffer NT 200 uL NT For gels containing gt 2 agarose double the volume of per Buffer NT The maximum amount of gel slice per NucleoSpin 100 mg gel Extract II Column is 400 mg or 200 mg of a high percentage gel 2 96 In this case 2 loading steps are required step 2 Incubate sample for 5 10 min at 50 C Vortex
15. l slots Remove the spin cup carefully from centrifuge and collection tube and avoid contact of spin cup with flow through Not enough elution buffer Especially when larger amounts of DNA 5 ug are bound increase elution buffer volume up to 100 uL Isolation of large DNA fragments Preheat Elution Buffer NE to 70 C and incubate on the silica membrane at room temperature for 2 min before centrifugation MACHEREY NAGEL 01 2011 Rev 11 19 PCR clean up Gel extraction Problem Possible cause and suggestions Appearance of additional bands on agarose gel Appearance of additional bands on agarose gel In case water is used for elution and agarose with a low ion content is used for agarose gel electrophoresis the formation of denaturated single stranded DNA might be promoted To re anneal the DNA add all components of the subsequent enzymatic reaction omitting the enzyme Incubate at 95 C for 2 min and let the mixture cool slowly to room temperature at this step the DNA re anneals Add the enzyme and continue with your downstream application Suboptimal performance of DNA in sequencing restriction or ligation reactions Carry over of ethanol ethanolic Buffer NT3 Centrifuge 5 min at 11 000 x g or better incubate column for 5 10 min at 70 C before elution to remove ethanolic Buffer NT3 completely Ethanolic contaminations are also indicated by gel loading problems samples float out
16. n of PCR products section 5 MACHEREY NAGEL 01 2011 Rev 11 17 NucleoSpin Extract II 7 2 Purification of single stranded DNA Buffer NTC The NucleoSpin Extract Il Buffer NT is able to bind single stranded DNA ssDNA gt 150 bases Shorter oligonucleotides especially primers are completely removed If you need to purify short ssDNA the additional Binding Buffer NTC can be used see Figure 3 Note Buffer NTC has to be ordered separately 100 mL Buffer NTC REF 740654 100 see ordering information Before starting the preparation Check if Wash Buffer NT3 was prepared according to section 3 Adjust DNA binding condition Mix 1 volume of sample with 2 volumes of Buffer NTC 2 vol NTC e g 100 uL PCR reaction mix and 200 uL Buffer NTC per 1 vol sample If your sample contains large amounts of detergents or other critical substances double the volume of Buffer NTC Bind DNA Continue with step 2 of the protocol for direct purification of PCR products section 5 1 2 3 e 490 bp m 490 bases 164 bp 164 bases 100 bases 64 bases 18 bases u NT NTC Figure 3 Purification of dsDNA and ssDNA using buffers NT and NTC PCR fragments amplified using one phosphorylated and one dephosphorylated primer were partially digested with A Exonuclease Samples were purified using Binding Buffer NT and NTC and run on a 1 TAE agarose gel Remaining double st
17. o removal of small fragments up to 100 bp Otherwise adding 1 to 3 volumes of water to 1 volume of Buffer NT will be sufficient Therefore for each size of small fragments gt 65 bp that has to be removed and for each PCR system you can determine the appropriate ratio of Buffer NT dilution in advance Figure 1 shows a purification result with a Buffer NT dilution series Pure Buffer NT lane 3 as well as Buffer NT plus one volume of water lane 4 lead to 100 recovery of a PCR fragment ladder lane 2 More diluted Buffer NT cuts off more and more of the low molecular mass bands Usually a dilution with 5 volumes of water should be sufficient to eliminate even larger unwanted primer dimer fragments while purifying the 164 bp fragment with gt 90 96 dilution 11 1 2 1 3 1 1 5 16 1 7 1 8 1 9 Figure 1 Purification of PCR reactions using Buffer NT dilutions Lane 1 GeneRuler 100 bp DNA Ladder MBI Fermentas Lane 2 DNA ladder input 21 base primer 50 65 79 100 164 359 645 and 982 bp fragment amplified using Biotaq DNA Polymerase Bioline Lane 3 Purification with 100 Buffer NT Lane 4 12 Purification with Buffer NT diluted with 1 9 volumes of water PCR is patented by Roche Diagnostics 8 MACHEREY NAGEL 01 2011 Rev 11 PCR clean up Gel extraction 2 4 Elution procedures For the elution of DNA one of the following solutions can be used Buffer NE supplied 5 mM Tris HCl pH 8 5 TE buffer pH 8
18. ol for PCR clean up 6 Protocol for DNA extraction from agarose gels 7 Support protocols 7 1 Purification of samples containing SDS Buffer NTB 7 2 Purification of single stranded DNA Buffer NTC 8 Appendix 8 1 Troubleshooting 8 2 Ordering information 8 3 References 8 4 Product use restriction warranty ana A A oN DD o 12 13 15 17 17 18 19 19 21 22 22 MACHEREY NAGEL 01 2011 Rev 11 PCR clean up Gel extraction 1 Components 1 1 Kit contents NucleoSpin Extract Il 10 preps 50 preps 250 preps REF 740609 10 740609 50 740609 250 Binding Buffer NT 10 mL 2x25mL 2x 120 mL Weeer 6 mL 2x6mL 40 mL Concentrate Elution Buffer NE 5mL 15 mL 50 mL in NucleoSpin Extract Il 10 50 250 Columns yellow rings Collection Tubes 2 mL 10 50 250 User Manual 1 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 4 MACHEREY NAGEL 01 2011 Rev 11 PCR clean up Gel extraction 1 2 Reagents consumables and equipment to be supplied by user Reagents 96 100 ethanol Consumables 1 5 mL microcentrifuge tubes Disposable pipette tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes Heating block Vortex mixer Personal protection equipment lab coat gloves goggles 1 3 About this User Manual It is strongly recommended reading the detailed protocol sections of this
19. r dimers or side products resulting from unspecific annealing since they might interfere with your downstream sequencing or cloning applications Removal of double stranded DNA gt 65 bp can be achieved by diluting an aliquot of Buffer NT with sterile water in an appropriate ratio and then proceeding with the standard protocol see section 5 Diluting Buffer NT in a certain range lowers the binding efficiency for small fragments without compromising the recovery of larger PCR products Which dilution ratio to choose depends on the fragment size that is to be purified as well as on the PCR buffer system that is used Influence of fragment size The smaller the fragment in question the less you have to dilute Buffer NT Influence of PCR buffer system The influence of the PCR buffer system on the removal of small fragments is more complex Some reaction buffers contain detergents like Tween or high concentrations of additives like betaine to lower the melting MACHEREY NAGEL 01 2011 Rev 11 T PCR clean up Gel extraction temperature of the DNA template These substances can usually be found in PCR buffers for high fidelity or long range PCR They tend to lower the binding efficiency of DNA to the silica membrane and therefore have to be considered when choosing a dilution ratio of Buffer NT As a rule of thumb if a PCR buffer system without special additives is used adding 3 to 5 volumes of water to 1 volume of Buffer NT will lead t
20. randed DNA can be seen as faint bands The corresponding single stranded DNA is running slightly faster due to secondary structure formation Compared to the input DNA u lane 1 Buffer NT removes ssDNA 150 bases NT lane 2 whereas Buffer NTC leads to full recovery of even primer oligonucleotides NTC lane 3 18 MACHEREY NAGEL 01 2011 Rev 11 PCR clean up Gel extraction 8 Appendix 8 1 Troubleshooting Problem Possible cause and suggestions Time and temperature Incomplete Check incubation temperature Depending on the weight of lysis of the gel slice incubation section 6 step 1 can be prolonged agarose up to 20 min Vortex every 2 min and check integrity of the gel slices slice Very large gel slices can be crushed before addition of Buffer NT Reagents not prepared properly Add indicated volume of 96 100 ethanol to Buffer NT3 Concentrate and mix well before use Incompletely dissolved gel slice Increase time or add another two volumes of Buffer NT and vortex the tube every 2 minutes during incubation at 50 C Small pieces of gel are hardly visible and contain DNA that will be lost for purification Insufficient drying of the NucleoSpin Extract II silica membrane Low Centrifuge 5 min at 11 000 x gor incubate column for 2 5 min at DNA yield 70 C before elution to remove ethanolic Buffer NT3 completely Ethanolic contaminations are also indicated by gel loading problems samples float out of ge
21. s performance NanoDrop Spectrophotometer technology is very sensitive of DNA in o to any particles included in the sample material To pellet the NanoDrop silica particles centrifuge gt 2 min at 11 000 x g and take the Spectro supernatant for further use photometer Analysis or Agilent s Bioanalyzer Carry over of chaotropic salts Low ratio Refer to detailed troubleshooting Suboptimal performance of Aseo Asi DNA in sequencing restriction or ligation reactions Carry over of chaotropic salts 8 2 Ordering information Product REF Pack of NucleoSpin Extract II 740609 10 50 250 10 50 250 Buffer NT 740614 100 100 mL Buffer NTB 740595 150 150 mL Buffer NTC 740654 100 100 mL aus 740588 20 mL Collection Tubes 2 mL 740600 1000 Visit www mn net com for more detailed product information MACHEREY NAGEL 01 2011 Rev 11 21 PCR clean up Gel extraction 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 8 4 Product use restriction warranty NucleoSpin Extract II 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
22. the sample 50 C briefly every 2 3 min until the gel slice is completely 5 10 min dissolved 2 Bind DNA Place a NucleoSpin Extract Il Column into a Collection Load sample Tube 2 mL and load the sample Centrifuge for 1 min at 11 000 x g Discard flow through 11 000 xg and place the column back into the collection tube 1 min MACHEREY NAGEL 01 2011 Rev 11 15 NucleoSpin Extract II Wash silica membrane Add 700 uL Buffer NT3 to the NucleoSpin Extract II Column Centrifuge for 1 min at 11 000 x g Discard flow amp 3 through and place the column back into the collection H 700 uL NT3 tube Note Carry over of chaotropic salt may result in low A JA 11 values To prevent problems in very sensitive downstream 000 xg applications or if the entire eluate has to be used follow the 1 min instructions given in section 8 1 Suboptimal performance of DNA in sequencing restriction or ligation reactions Carry over of chaotropic salts Dry silica membrane completely Make sure the spin column does not come in contact with the flow through while removing it from the Centrifuge for 2 min at 11 000 x gto remove Buffer NT3 D centrifuge and the collection tube Note Residual ethanol from Buffer NT3 might inhibit 11 000 x g enzymatic reactions Total removal of ethanol can be 2 min achieved by incubating the columns for 2 5 min at 70 C prior to elution Elute DNA 15 50 uL NE Place the Nu
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