PCR clean-up Gel extraction
Contents
1. Before starting any NucleoSpin Extract II protocol prepare the following e Add the indicated volume of 96 100 ethanol to buffer NT3 concentrate 10 preps 50 preps 250 preps Cat No 740609 10 740609 50 740609 250 7 ml 2x7 ml 40 ml Buffer NT3 add 28 ml ethanol add 28 ml ethanol add 160 ml ethanol 10 MACHEREY NAGEL 09 2005 Rev 02 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 NT nidine 7 Xn Harmful by inhalation in contact R 20 21 22 S 13 onan 96 7 with skin and if swallowed Risk Phrases R 20 21 22 Harmful by inhalation in contact with the skin and if swallowed Safety Phrases 13 Keep away from food drink and animal feedstuffs Label not necessary if quantity below 125 g or ml concerning 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 42 and TRGS 200 7 1 MACHEREY NAGEL 09 2005 Rev 02 11 NucleoSpin Extract II 5 Protocol for DNA extraction from agarose gels Excise DNA fragment 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 Determine the weight of the gel slice and transfer it to a clean tube Gel lysis For each 100 mg of agarose gel add 200 pl buffer2. PCR clean up Gel extraction User manual NucleoSpin Extract II September 2005 Rev 02 MACHEREY NAGEL MN Protocol at a glance Rev 02 NucleoSpin Extract II Gel extraction PCR clean up 1 Excise DNA fragment Z 2 Gel lysis 200 pI NT Adjust binding conditions 100 mg 200 pI NT 100 pl 50 C 5 10 min 3 Bind DNA 1 min 11 000 x g 4 Wash silica membrane 600 uI NT3 1 min 11 000 x g 5 Dry silica membrane 2 min 11 000 x g 6 Elute DNA 15 50 pl NE 1 min RT 1 min 11 000 x g PCR clean up Gel extraction Table of contents 1 Kit contents 2 Product description 2 1 2 2 2 3 2 4 2 5 The basic principle About this user manual Kit specifications Removal of small DNA fragments and primer dimers Elution procedures 3 Storage conditions and preparation of working solutions 4 Safety instructions risk and safety phrases 5 Protocol for DNA extraction from agarose gels 6 Protocol for direct purification of PCR products 7 Support protocols YA 7 2 7 3 Concentration and removal of salts enzymes etc Purification of samples without SDS buffer NT Concentration and removal of salts enzymes etc Purification of samples containing SDS buffer NTB Purification of single stranded DNA buffer NTC 8 Appendix 8 1 8 2 8 3 8 4 MACHEREY NAGEL 09 2005 Rev 02 Troubleshooting Ordering information References Pro
3. 10 kb the use of our NucleoTraP CR kit is recommended Table 1 DNA Recovery with NucleoSpin Extract II Fragment length Elution volume Recovery 15 pl 85 65 bp 25 ul 90 50 pl 95 100 pl 95 15 ul 85 400 bp 25 ul 95 Yo 50 pl 100 Yo 100 pl 100 Yo 15 ul 85 700 bp 25 ul 90 Yo 50 pl 95 100 pl 95 15 ul 85 1500 bp 25 ul 85 Yo 50 ul 90 100 pl 95 Yo PCR is patented by Roche Diagnostics 8 MACHEREY NAGEL 09 2005 Rev 02 PCR clean up Gel extraction Reference ladder Elution volume pl 100 75 50 25115 15 15 15 20 20 20 20 25 25 25 25 mm ee ee G kad ad ad kad eed ee eed ee Recovery 75 lt ____ _ _ _ gt 100 Figure 2 DNA recovery with different elution volumes A PCR sample with a fragment size of 782 bp was purified from a 1 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 pl loading dye For analysis the mixture was loaded on a 1 TAE gel The recovery was estimated by comparison with a fragment ladder MACHEREY NAGEL 09 2005 Rev 02 9 PCR clean up Gel extraction 3 Storage conditions and preparation of working solutions Attention Buffer NT contains chaotropic salt Wear gloves and goggles e The NucleoSpin Extract II kit should be stored at room temperature and is stable for up to one year
4. II columns for 2 5 min at 70 C prior to elution Elute DNA Place the NucleoSpin Extract Il column into a clean 1 5 ml microcentrifuge tube Add 15 50 ul elution buffer NE and incubate at room temperature for 1 min to increase the yield of eluted DNA Centrifuge for 1min at 11 000 x g Yield of larger fragments gt 5 10 kb can be increased by using prewarmed elution buffer 70 C For elution add prewarmed elution buffer and incubate at room temperature for 1 min before collecting eluate by centrifugation MACHEREY NAGEL 09 2005 Rev 02 2 min 11 000 xg 15 50 pl NE 1 min 11 000 x g 13 NucleoSpin Extract II e2 Protocol for direct purification of PCR products Adjust DNA binding conditions Mix 1 volume of sample with 2 volumes of buffer NT e g mix 100 pl PCR reaction and 200 ul NT For sample volumes lt 50 ul adjust the volume of the reaction mix to 50 ul using TE buffer pH 7 5 For removal of DNA fragments lt 65 bp dilutions of buffer NT can be used instead of 100 NT Please refer to section 2 4 Bind DNA Place a NucleoSpin Extract II column into a 2 ml collecting tube and load the sample Centrifuge for 1 min at 11 000 x g Discard flow through and place the NucleoSpin Extract ll column back into the collecting tube Wash silica membrane Add 600 ul buffer NT3 Centrifuge for 1 min at 11 000 x g Discard flow through and place the NucleoSpin Extract
5. High amount of agarose e Use doubled volumes of buffer NT for highly concentrated a o LMP low melting point agarose gels Incomplete lysis of Time and temperature agarose e Check incubation temperature Depending on the weight of the slices gel slice incubation section 5 step 2 can be prolonged up to 20 min Vortex every 2 min and check integrity of the gel slice Very large gel slices can be quenched or crushed before addition of buffer NT Reagents not applied properly e Add indicated volume of 96 100 ethanol to buffer NT3 concentrate and mix well before use Incompletely dissolved gel slice e Increase time or add another two volumes of 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 DNA e Centrifuge 5 min at 11 000 x g or incubate column for 2 5 min at yield 70 C before elution to remove ethanolic buffer NT3 completely Ethanolic contaminations are also indicated by gel loading problems samples float out of gel slots Remove the spin cup carefully from centrifuge and collecting tube and avoid contact of spin cup with flow through Not enough elution buffer e Especially when larger amounts of DNA gt 5 ug are bound increase elution buffer volume up to 100 ul Isolation of large DNA fragments e Preheat elution buffer NE to 70 C and incubate on t
6. Il column back into the collecting tube Dry silica membrane Centrifuge for 2 min at 11 000 x g to remove buffer NT3 quantitatively Make sure the spin column doesn t come in contact with the flow through while removing it from the centrifuge and the collecting tube Residual ethanol from buffer NT3 might inhibit subsequent reactions and has to be removed in this step In addition to centrifugation total removal can be achieved by incubation of NucleoSpin Extract II columns for 2 5 min at 70 C prior to elution 2 vol NT per 1 vol sample load sample 1 min 11 000 xg 600 pI NT3 1 min 11 000 xg 2 min 11 000 xg MACHEREY NAGEL 09 2005 Rev 02 NucleoSpin Extract II Elute DNA Place the NucleoSpin Extract II column into a clean prewarmed elution buffer 70 C For elution add prewarmed 11 000 elution buffer and incubate at room temperature for 1 min d xg before collecting eluate by centrifugation 1 5 ml microcentrifuge tube Add 15 50 pl elution buffer 15 50 pl NE NE and incubate at room temperature for 1 min to increase the yield of eluted DNA Centrifuge for 1min at 11 000 x g Yield of larger fragments gt 5 10 kb can be increased by using e gt 1 min MACHEREY NAGEL 09 2005 Rev 02 15 NucleoSpin Extract II 7 Support protocols 7 1 Concentration and removal of salts enzymes etc Purification of samples without SDS buffer NT Note Buffer NT i
7. NT For gels containing gt 2 agarose double the volume of buffer NT The maximum amount of gel slice per NucleoSpin Extract Il column is 400 mg or 200 mg of a high percentage gel gt 2 In this case 2 loading steps are required step 3 Incubate sample at 50 C until the gel slices are dissolved 5 10 min Vortex the sample briefly every 2 3 min until the gel slices are dissolved completely Bind DNA Place a NucleoSpin Extract II column into a 2 ml collecting tube and load the sample Centrifuge for 1 min at 11 000 x g Discard flow through and place the NucleoSpin Extract ll column back into the collecting tube Wash silica membrane Add 600 ul buffer NT3 Centrifuge for 1 min at 11 000 x g Discard flow through and place the NucleoSpin Extract Il column back into the collecting tube MACHEREY NAGEL 09 2005 Rev 02 200 pl NT per 100 mg gel 50 C 5 10 min load sample 1 min 11 000 xg 600 pI NT3 1 min 11 000 xg NucleoSpin Extract II Dry silica membrane Centrifuge for 2 min at 11 000 x g to remove buffer NT3 quantitatively Make sure the spin column doesn t come in contact with the flow through while removing it from the centrifuge and the collecting tube Residual ethanol from buffer NT3 might inhibit subsequent reactions and has to be removed in this step In addition to centrifugation total removal can be achieved by incubation of NucleoSpin Extract
8. al purification of DNA from agarose Proc Natl Acad Sci USA 76 615 619 8 4 Product use restriction warranty NucleoSpin Extract ll kit components were developed designed and sold for research purposes only They are suitable for in vitro uses only No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoSpin Extract II kit for a specific application range as the performance characteristic of this kit has not been verified to a specific organism 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 an extra copy MACHEREY NAGEL does not warrant against 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 against defects in products or components not manufactured by MACHEREY NAGEL or against damages res
9. dder input 21 b primer 50 65 79 100 164 359 645 and 982 bp fragment amplified using Biotaq DNA Polymerase Bioline Lane 3 Purification with 100 NT Lane 4 12 Purification with NT diluted with 1 9 volumes of water MACHEREY NAGEL 09 2005 Rev 02 7 PCR clean up Gel extraction 2 5 Elution procedures For elution of DNA one of the following solutions can be used Buffer NE supplied 5 mM Tris HCI pH 8 5 TE buffer pH 8 5 distilled water pH 8 5 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 yields See Table 1 for the correlation between dispensed elution buffer volumes and typical recoveries for 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 1 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 kb can be increased by using prewarmed elution buffer 70 C For elution add prewarmed elution buffer NE and incubate for 1 2 min before collecting eluate by centrifugation For fragments gt
10. duct use restriction warranty 10 11 12 14 16 16 16 17 18 18 19 20 20 PCR clean up Gel extraction 1 Kit contents 10 preps 740609 10 50 preps 740609 50 250 preps 740609 250 Buffer NT 6 ml 30 ml 2x75 ml Buffer NT3 concentrate 7 ml 2x7ml 40 ml Buffer NE 5 ml 15 ml 50 ml in NucleoSpin Extract II 10 50 250 columns yellow NucleoSpin collecting tubes 2 ml La 20 230 Protocol 1 1 1 4 MACHEREY NAGEL 09 2005 Rev 02 PCR clean up Gel extraction 2 Product description 2 1 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 II columns Contaminations like salts and soluble macromolecular components are removed by a simple washing step with ethanolic buffer NT3 Pure DNA is finally eluted under low ionic strength conditions with slightly alkaline buffer NE 5 mM Tris HCl pH 8 5 2 2 About this user manual Experienced users who are performing the purification of PCR products or DNA fragments from agarose gels using a NucleoSpin Extract II isolation kit may refer to the Protocol at a glance instead of this user manual The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure First time users are strong
11. ecially 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 NTC Ref 740654 100 see Ordering information 1 Adjust DNA binding conditions Mix 1 volume of sample with 2 volumes of buffer NTC e g 100 pl PCR reaction mix and 200 pl NTC per 2 vol NTC 1 vol sample If your sample contains large amounts of detergents or other critical substances double the volume of NTC 2 Bind DNA Continue with step 2 of the protocol for direct purification of PCR products section 6 bp a Ran bases bp bases bases bases bases 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 l Exonuclease Samples were purified using binding buffers NT and NTC and run on a 1 TAE agarose gel Remaining double stranded 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 NT removes ssDNA lt 150 bases NT lane 2 whereas NTC leads to full recovery of even primer oligonucleotides NTC lane 3 MACHEREY NAGEL 09 2005 Rev 02 17 PCR clean up Gel extraction 8 Appendix 8 1 Troubleshooting Problem Possible cause and suggestions
12. he silica membrane at room temperature for 2 min before centrifugation MACHEREY NAGEL 09 2005 Rev 02 PCR clean up Gel extraction Problem Possible cause and suggestions Carry over of ethanol ethanolic buffer NT3 e Centrifuge 5 min at 11 000 x g or incubate column for 2 5 min at 70 C before elution to remove ethanolic buffer NT3 completely Ethanolic contaminations are also indicated by gel loading problems samples float out of gel slots Remove the spin cup carefully from centrifuge and collecting tube and avoid contact of Suboptimal spin cup with flow through performance of DNA in Elution of DNA with buffers other than buffer NE e g TE buffer sequencing Tris EDTA reactions e 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 e Quantify DNA by agarose gel electrophoresis before setting up sequencing reactions 8 2 Ordering information Product Cat No Pack of NucleoSpin Extract II 740609 10 10 preps NucleoSpin Extract II 740609 50 50 preps NucleoSpin Extract II 740609 250 250 preps Buffer NT 740614 100 100 ml Buffer NTB 740595 150 150 ml Buffer NTC 740654 100 100 ml one 740506 zom NucleoSpin collecting tubes 740600 1000 2 ml MACHEREY NAGEL 09 2005 Rev 02 19 PCR clean up Gel extraction 8 3 References Vogelstein B and D Gillespie 1979 Preparative and analytic
13. ly advised to read the entire user manual 2 3 Kit specifications e The NucleoSpin Extract II kit is designed for the purification of DNA from TAE TBE agarose gels and for direct purification of PCR products two applications in one kit e The NucleoSpin Extract ll buffer formulation ensures complete removal of primers from PCR reactions while small DNA fragments are still bound and purified with high recovery e With NucleoSpin Extract Il even DNA fragments from PCR reaction buffers rich in various detergents can be purified with high recovery e The adsorption of DNA to the NucleoSpin Extract Il membrane is pH dependent Optimal recovery is achieved by using TAE standard gels or reaction mixtures with pH 6 8 e Standard as well as low melting agarose gels can be used e The prepared DNA fragments can be used directly in applications like automated fluorescent DNA sequencing PCR or any kind of enzymatic manipulation PCR is patented by Roche Diagnostics MACHEREY NAGEL 09 2005 Rev 02 5 PCR clean up Gel extraction Kit specification at a glance Parameters NucleoSpin Extract II DNA fragments from agarose gels v Concentration removal of salts enzymes nucleotides a o labeling reagents like biotin or radioactive u ATP etc Direct purification of amplified DNA v Elution volume 15 50 ul Binding capacity 15 ug Time prep a Purification of reaction mixtures without SDS see sectio
14. n 7 1 Purification of reaction mixtures asee containing SD Purification of single stranded DNA see section 7 3 Removal of small DNA fragments see section 2 4 and primer dimers 2 4 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 primer dimers or side products resulting from unspecific annealing since they might interfere with your downstream sequencing or cloning applications Removal of double stranded DNA lt 65 bp can be achieved by diluting an aliquot of buffer NT with sterile water in an appropriate ratio and then proceed with the standard protocol see section 6 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 is the less you have to dilute buffer NT 6 MACHEREY NAGEL 09 2005 Rev 02 PCR clean up Gel extraction 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
15. of additives like betaine to lower the melting 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 to 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 So for each size of small fragments lt 65 bp that has to be removed and for each PCR system there may be checked the appropriate ratio of buffer NT dilution in advance Figure 1 shows a purification result with an NT dilution series Pure NT lane 3 as well as NT plus one volume of water lane 4 lead to 100 recovery of a PCR fragment ladder lane 2 Use of 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 bp 982 AAA AAA 645 359 LITI J N _ dilution factor 111 1 2 13 1 4 1 5 1 6 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 la
16. s provided with the NucleoSpin Extract II kits 1 Adjust DNA binding conditions Mix 1 volume of sample with 2 volumes of buffer NT 2 vol NT e g 200 uI NT and 100 ul reaction mixture per 1 vol sample If your sample contains large amounts of detergents or other critical substances double the volume of NT 2 Bind DNA Continue with step 2 of the protocol for direct purification of PCR products section 6 7 2 Concentration and removal of salts enzymes etc Purification of samples containing SDS buffer NTB The NucleoSpin Extract ll buffer NT is compatible with most commonly used detergents except sodium dodecyl sulfate SDS For purification of DNA from SDS containing buffers e g 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 NTB Ref 740595 150 see Ordering information 1 Adjust DNA binding conditions 5 vol NTB Mix 1 volume of sample with 5 volumes of buffer NTB per e g 100 pl reaction with 500 pl NTB 1 vol sample 2 Bind DNA Continue with step 2 of the protocol for direct purification of PCR products section 6 16 MACHEREY NAGEL 09 2005 Rev 02 NucleoSpin Extract II 7 3 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 esp
17. ulting 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 20 MACHEREY NAGEL 09 2005 Rev 02 PCR clean up Gel extraction 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
18. 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 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 Please contact MACHEREY NAGEL Germany Tel 49 2421 969 270 e mail TECH BlIO mn net com MACHEREY NAGEL 09 2005 Rev 02 21
Download Pdf Manuals
Related Search