PCR clean-up Gel extraction
Contents
1. MACHEREY NAGEL 01 2012 Rev 02 19 NucleoSpin Gel and PCR Clean up 5 2 DNA extraction from agarose gels Before starting the preparation Check if Wash Buffer NT3 was prepared according to section 3 Excise DNA fragment solubilize gel slice Note Minimize UV exposure time to avoid damaging the DNA Refer to section 2 5 for more tips on agarose gel extraction Take a clean scalpel to excise the DNA fragment from an agarose gel Remove all excess agarose Determine the weight of the gel slice and transfer it to a clean tube For each 100 mg of agarose gel lt 2 add 200 pL 200 pL NTI Buffer NTI per For gels containing gt 2 agarose double the volume of 100 mg gel Buffer NTI Incubate sample for 5 10 min at 50 C Vortex the sample briefly every 2 3 min until the gel slice is completely 50 C dissolved 5 10 min 2 Bind DNA Place a NucleoSpin Gel and PCR Clean up Column into a Collection Tube 2 mL and load up to 700 uL Load sample sample Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the collection tube 11 aad xg s Load remaining sample if necessary and repeat the centrifugation step 3 Wash silica membrane 700 uL NT3 Add 700 uL Buffer NT3 to the NucleoSpin Gel and PCR Clean up Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the 11 000 x g collection tube 30 f 20 MACHEREY NA2. PCR clean up Gel extraction User manual NucleoSpin Gel and PCR Clean up January 2012 Rev 02 MACHEREY NAGEL MN PCR clean up gel extraction Protocol at a glance Rev 02 PCR Gel DNA Single clean up extraction clean up stranded with SDS DNA clean up 1 PCR clean up DNA clean up or single stranded DNA clean up Adjust binding condition Gel extraction Excise DNA fragment solubilize gel slice 200 uL NTI 200 uL NTI 500 uL NTB 200 uL NTC 100 uL PCR 100 mg gel 100 uL sample 100 uL sample 50 C 5 10 min 2 Bind DNA e gt 11 000 x g 30s 3 Wash silica membrane 700 uL NT3 11 000 x g 30s Recommended 2nd wash 700 uL NT3 11 000 x g 30s 4 Dry silica membrane e gt 11 000 x g 1 min 5 Elute DNA 15 30 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 Components 1 1 Kit contents 1 2 Reagents consumables and equipment to be supplied by user 1 3 About this user manual Product description 2 1 The basic principle 2 2 Kit specifications 2 3 Removal of small DNA fragments and primer dimers 2 4 pH indicator 2 5 Tips and tricks for extractions from agarose gels 2 6 DNArecovery depends on fragment size an
3. DNA Polyacrylamide gels are usually extracted by the crush and soak method where a small piece of gel is crushed and incubated in a diffusion buffer The DNA is then allowed to passively diffuse out of the gel and is then purified from the diffusion buffer The diffusion buffer 500 mM ammonium acetate pH 8 0 0 1 SDS 1 mM EDTA 10 mM magnesium acetate is not provided with the kit 1 Prepare sample Excise the DNA fragment with a scalpel or razor blade Excise DNA in a minimal amount of polyacrylamide Weigh the gel fragment slice and transfer it to a 1 5 ml microcentrifuge tube not provided 2 Crush gel Crush the gel slice using a disposable pipette tip with a Crush gel melted end to resemble a pestle for the microcentrifuge slice tube mortar The smaller the pieces the better the DNA recovery 3 Extract DNA 50 C 30 60 min Add 200 uL of diffusion buffer to each 100 mg of crushed gel Make sure that all gel pieces are submerged or in diffusion buffer 37 C Incubate for 30 60 min at 50 C or over night at 37 C over night 4 Remove polyacrylamide 11 000 x g 1 min Centrifuge for 1 min at 14 000 x g to pellet the polyacryl amide and transfer the supernatant to a new microcentri Transfer fuge tube not provided supernatant Alternatively transfer the mixture to a NucleoSpin Gel or and PCR Clean up Column and centrifuge 1 min at 11 000 14 000 x g to retain the gel on the column Keep the flow
4. 6 MACHEREY NAGEL 01 2012 Rev 02 PCR clean up gel extraction The cut off for small DNA fragments can be shifted from lt 50 bp to several hundred bp by diluting Binding Buffer NTI to remove primer dimers from target PCR products details in section 2 3 The pH indicator in Binding Buffer NTI ensures optimal binding conditions with pH lt 7 0 details in section 2 4 The yellow color makes it easier to identify undissolved agarose during DNA gel extraction NucleoSpin Gel and PCR Clean up can be used with all kinds of agarose gels high or low melting with 1 to 5 agarose and a variety of buffer systems like TAE or TBE tips and tricks in section 2 5 The kit also works with low conductivity borate electrophoresis systems Concentrated elution in down to 10 uL Elution Buffer NE details in section 2 6 Several support protocols extend the application range of NucleoSpin Gel and PCR Clean up to Clean up of DNA from reaction mixtures containing SDS section 5 5 Clean up of single stranded DNA section 5 6 Extraction of RNA from agarose gels section 5 4 Extraction of DNA from polyacrylamide gels section 5 3 The purified and concentrated DNA can directly be used for hybridization sequencing PCR restriction ligation in vitro transcription labeling or any other kind of enzymatic reaction Table 1 Kit specifications at a glance Parameter NucleoSpin Gel and PCR Clean up Sample materi
5. ethanol to reconstitute Buffer NT3 or ethanol that is not denatured The denaturing components may not evaporate as fast as ethanol and end up concentrated in the eluate inhibiting enzymes like ligase Carry over of chaotropic salts Perform the optional washing step Additionally 250 uL NT3 can be loaded before the drying step Note The volume of Buffer NT3 included in the kit is not sufficient for this modification for all preparations but can be ordered separately see ordering information 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 28 MACHEREY NAGEL 01 2012 Rev 02 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 particles 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 Sp
6. oligonucleotides NTC lane 3 26 MACHEREY NAGEL 01 2012 Rev 02 PCR clean up gel extraction 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Time and temperature Incomplete Check incubation temperature and volume of Buffer NTI dissolving of Increase incubation time Vortex every 2 min and check integrity gel slice of the gel slice Very large gel slices can be crushed before addition of Buffer NTI to shorten the melting time 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 NTI 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 Gel and PCR Clean up Low silica membrane DNA yield 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 the centrifuge and collection tube and avoid contact of spin cup with flow through Incomplete elution Especially for larger amounts of DNA gt 5ug long DNA fragments gt 1000 bp or after gel extraction do multiple elution steps with fresh bu
7. 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 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 Trademarks NanoDrop is a registered trademark of NanoDrop Technologies Inc NucleoSpin is a registered trademark of MACHEREY NAGEL GmbH amp Co KG Roti is a registered trademark of Carl Roth 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 32 MACHEREY NAGEL 01 2012 Rev 02
8. 100 7 3 016 1 2 0 14 u 80 7 Ss 0 124 S j amp 010 amp 60 E J 0 08 a 40 g 0 06 5 zZ uJ a 0 04 20 7 lt 4 4 0 02 A 0 0 1 1 0 7 7 7 r Sub 10pL 15pL 20L 25L 30 pL Sub 10pL 15pL 20L 25L 30 pL Elution volume Elution volume Figure 5 Elution volume dependent DNA recovery and concentration 2 ug of 100 bp DNA ladder Fermentas were purified from standard PCR buffer and eluted with increasing volumes 2 7 Salt carry over and low A A The silica membrane technology to purify RNA or DNA is based on the ability of chaotropic salts to destroy the water shell around nucleic acids Two commonly used chaotropic salts are guanidine hydrochloride GUHCI and guanidinium thiocyanate GuSCN In solution they both have the same guanidinium cation but different anions These anions are not only responsible for their different behavior towards nucleic acids but also for their different UV absorption spectra GUHCI exhibits only minimal absorption lt 220 nm even at a concentration of 1 M whereas GuSCN already shows significant absorption lt 240 nm 1 mM Figure 6 and even lt 260 nm 1 M 1 0 0 8 En 0 6 0 4 Absorption 0 2 4 04 200 210 220 230 240 250 260 270 280 290 300 Wave length nm Figure 6 UV absorption spectra of 1 mM GuHCI solid line and 1 mM GuSCN dotted line Especially the difference in absorption at 230 nm can have a huge impact on the p
9. Buffer NT3 Finally the pure DNA is eluted under low salt conditions with slightly alkaline Elution Buffer NE 5 mM Tris HCl pH 8 5 2 2 Kit specifications NucleoSpin Gel and PCR Clean up is designed for fast purification of PCR products such as DNA from enzymatic reactions as well as the extraction of DNA fragments from TAE or TBE agarose gels Only two volumes of binding buffer per volume of sample are needed to process up to 200 uL of PCR enzymatic reaction or 200 mg of agarose gel with only one loading step By adding additional Binding Buffer NTI see ordering information it is possible to load an unlimited amount of sample volumes onto a single column tips and tricks in section 2 5 Up to 15 ug DNA from 50 bp to at least 20 kbp can be purified efficiently in 10 20 min with average recoveries from 60 to 90 depending on the fragment size and elution procedure details in section 2 6 The NucleoSpin Gel and PCR Clean up buffer formulation ensures complete removal of all kinds of contaminations such as nucleotides primers enzymes mineral oil PCR additives e g salts betaine DMSO detergents e g Tween 20 Triton X 100 dyes e g ethidiumbromide crystal violet Stain G Midori Green Roti Safe GelStain unbound labels and tags Primers from PCR reactions are quantitatively eliminated while small DNA fragments are still bound and purified with high recovery details in section 2
10. 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
11. GEL 01 2012 Rev 02 NucleoSpin Gel and PCR Clean up Recommended Repeat previous washing step to minimize chaotropic salt carry over and low A A see section 2 7 for detailed information z F00 PENTA 11 000 x g 30 s Dry silica membrane Centrifuge for 1 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 inhibit ES 11 000 x g enzymatic reactions Total removal of ethanol can be 1 min achieved by incubating the columns for 2 5 min at 70 C prior to elution Elute DNA Place the NucleoSpin Gel and PCR Clean up Column Q 15 30 pL NE into a new 1 5 mL microcentrifuge tube not provided Add 15 30 uL Buffer NE and incubate at room RT temperature 18 25 C for 1 min Centrifuge for 1 min 1 min at 11 000 x g Note DNA recovery of larger fragments gt 1000 bp can be C5 u N es increased by multiple elution steps with fresh buffer heating 1 min to 70 C and incubation for 5 min See section 2 6 for detailed information MACHEREY NAGEL 01 2012 Rev 02 21 NucleoSpin Gel and PCR Clean up 5 3 DNA extraction from polyacrylamide gels In polyacrylamide gels the acrylamide monomers are covalently linked in a chemical reaction Therefore the gel cannot be dissolved like agarose gels to extract the trapped
12. 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 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 t
13. The smaller the fragment in question the less you have to dilute Buffer NTI 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 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 NTI 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 NTI will lead to removal of small fragments up to 100 bp Otherwise adding 1 to 3 volumes of water to 1 volume of Buffer NTI will be sufficient Therefore for each size of small fragments gt 50 bp that has to be removed and for each PCR system you can determine the appropriate ratio of Buffer NTI dilution in advance Figure 1 shows a purification result with a Buffer NTI dilution series Pure Buffer NTI lane 3 as well as Buffer NTI plus one volume of water lane 4 lead to 100 recovery of a PCR fragment ladder lane 2 More diluted Buffer NTI 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
14. al Up to 200 uL of PCR reaction or 200 mg of gel more sample with additional Binding Buffer NTI and multiple loading steps Binding capacity 25 ug Fragment length 50 bp 20 kbp Elution volume 10 30 uL Optimal recovery lt 15 ug 100 500 bp 30 uL Preparation time 10 min for 6 PCR purifications 20 min for 6 gel extractions MACHEREY NAGEL 01 2012 Rev 02 T PCR clean up gel extraction 2 3 Removal of small DNA fragments and primer dimers NucleoSpin Gel and PCR Clean up is designed to remove even traces of unused primer while purifying PCR products down to 50 bp at the same time However in some cases it is necessary to exclude these small fragments For example primer dimers or side products resulting from unspecific annealing may interfere with downstream sequencing or cloning applications Removal of double stranded DNA gt 50 bp can be achieved by diluting an aliquot of Buffer NTI with sterile water in an appropriate ratio and then proceeding with the standard protocol see section 5 1 Diluting Buffer NTI in a certain range lowers the binding efficiency for small fragments without compromising the recovery of larger PCR products However the dilution ratio will highly depend on the fragment Therefore for each size of small fragments gt 50 bp that has to be removed as well as for each PCR system the appropriate ratio of Buffer NTI dilution can be determined in advance Influence of fragment size
15. d elution volume 2 7 Salt carry over and low A A Storage conditions and preparation of working solutions Safety instructions 4 1 Risk and safety phrases 4 2 GHS classification Protocols 5 1 PCR clean up 5 2 DNA extraction from agarose gels 5 3 DNA extraction from polyacrylamide gels 5 4 RNA extraction from agarose gels Buffer NTC 5 5 DNA clean up of samples containing SDS Buffer NTB 5 6 Single stranded DNA clean up Buffer NTC Appendix 6 1 Troubleshooting 6 2 Ordering information 6 3 References 6 4 Product use restriction warranty 15 16 16 17 18 18 20 22 24 25 26 27 27 30 30 31 MACHEREY NAGEL 01 2012 Rev 02 PCR clean up gel extraction 1 Components 1 1 Kit contents NucleoSpin Gel and PCR Clean up 10 preps 50 preps 250 preps REF 740609 10 740609 50 740609 250 Binding Buffer NTI 10 mL 2x25mL 2x 120 mL Wash Burer NTS 6 mL 20 mL 2x 50 mL Concentrate Elution Buffer NE 5 mL 15 mL 50 mL NucleoSpin Gel and PCR Clean up Columns yellow 10 50 250 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 2012 Rev 02 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 Dis
16. die Umwelt vermeiden Besondere Anweisungen einholen Sicherheitsdaten blatt zu Rate ziehen 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 20 3 and TRGS 200 7 1 For further information see Material Safety Data Sheet 16 MACHEREY NAGEL 01 2012 Rev 02 PCR clean up gel extraction 4 2 GHS classification Only harmful features must not 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 Gefahrstoff GHS Symbol H S tze P S tze NTI Guanidinium thiocyanate Warning 302 412 260 273 30 60 EUH031 301 312 Guanidiniumthiocyanat 30 60 Achtung 330 Hazard phrases H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 412 Harmful to aquatic life with long lasting effects Sch dlich f r Wasserorganismen mit langfristiger Wirkung EUH 031 Contact with acids liberates toxic gas Entwickelt bei Ber hrung mit S ure giftige S ure Precaution phrases P 260 Do not breathe vapours Dampf nicht einatmen P 272 Contaminated work clothing should not be allowed out of the workplace Freisetzung in die Umwelt vermeiden P 301 312 IF SWALLOWED Call a POISON CENTER or docto
17. e rest stop the gel and cut out the band Expose the gel to UV light as short as possible Use the longest UV wave length that is allowed by your gel documentation system Prolonged exposure and short wave lengths can damage the DNA Wear gloves and a face mask to protect your skin and eyes from UV light Make sure to cut through the gel vertically and remove all excess agarose Use 0 7 1 0 agarose gels rather than higher percentages Make sure to actually weigh the gel since its weight is easily underestimated Up to 200 mg of agarose gel can be dissolved with 400 uL of Buffer NTI and loaded onto the column in one step However virtually unlimited amounts of gel can be loaded without clogging the column by increasing Buffer NTI proportionally see ordering information and adding multiple loading steps 10 MACHEREY NAGEL 01 2012 Rev 02 PCR clean up gel extraction 2 6 DNA recovery depends on fragment size and elution volume Upon completion of the wash steps with Buffer NT3 the DNA will adhere to the silica membrane The number of interactions with Si OH groups of the silica increase with the size of the DNA fragment As a result large DNA with several kilo base pairs binds much stronger and is much more difficult to elute than small DNA with just several hundred base pairs NucleoSpin Gel and PCR Clean up is recommended for DNA up 10 15 kbp Longer fragments can be purified but recovery may be low In addition fragme
18. ectro 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 Asso DNA in sequencing restriction or ligation reactions Carry over of chaotropic salts and see section 2 7 for detailed information MACHEREY NAGEL 01 2012 Rev 02 29 PCR clean up gel extraction 6 2 Ordering information Product REF Pack of NucleoSpin Gel and PCR Clean up 740609 10 50 250 10 50 250 Buffer NTI 740305 120 120 mL Buffer NTB 740595 150 150 mL Buffer NTC 740654 100 100 mL ma 740598 20 mL Collection Tubes 2 mL 740600 1000 NucleoTrap 740584 10 50 250 10 50 250 NucleoTrap CR 740587 10 50 250 10 50 250 Visit www mn net com for more detailed product information 6 3 References Vogelstein B and D Gillespie 1979 Preparative and analytical purification of DNA from agarose Proc Natl Acad Sci USA 76 615 619 30 MACHEREY NAGEL 01 2012 Rev 02 PCR clean up gel extraction 6 4 Product use restriction warranty NucleoSpin Gel and PCR Clean up 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
19. ffer heat to 70 C and incubate for 5 min See section 2 6 for detailed information MACHEREY NAGEL 01 2012 Rev 02 27 PCR clean up gel extraction Problem Possible cause and suggestions Appearance of additional bands on agarose gel after gel extraction DNA was denatured during purification In case where water is used for elution or 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 Use fresh running buffer and run at low voltage to lower the temperature High temperature might promote DNAdenaturation during electrophoresis Suboptimal performance of DNA in sequencing restriction or ligation reactions Carry over of ethanol ethanolic Buffer NT3 Before elution centrifuge 5 min at 11 000 xg or incubate column for 5 10 min at 70 C 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 the centrifuge and collection tube without having the spin cup make contact with the flow through Use either a different brand of
20. gel extraction is 10 20 less efficient than elution of purified PCR products In addition elution of several kbp long DNA fragments is 10 30 less efficient than elution of 500 bp fragments To improve the DNA recovery after gel extraction and or for large DNA fragments the following modifications can be applied to the standard elution procedure Heat elution buffer to 70 C and incubate elution buffer on the column at 70 C for 5 minutes Apply elution buffer to the column and centrifuge first at 30 50 x g for 1 min and then at 11 000 x g for 1 min Do 2 or better 3 elution steps with 20 or 30 uL fresh elution buffer Figure 4 demonstrates the increase in recovery for a 5 kbp fragment by 20 30 100 7 80 4 60 aul 20 uL 40 E 30 pL 20 0 T 1 1x 2x 3x Elution repeats DNA recovery Figure 4 Multiple elution steps increase recovery 3 ug of a 5 kbp fragment were purified from standard PCR buffer and eluted 1 2 or 3 times with 20 or 30 uL of fresh elution buffer If higher DNA concentrations are required elution volumes lt 30 uL can be used Keep in mind that although the concentration can be more than doubled Figure 5 A total DNA recovery will be significantly reduced for volumes lt 15 uL Figure 5 B For large DNA fragments and results after gel extraction the losses may be even more pronounced 12 MACHEREY NAGEL 01 2012 Rev 02 PCR clean up gel extraction A B 0 187
21. he 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 MACHEREY NAGEL is not liable for damages or defects arising in shipping and handling transport insurance for customers excluded or MACHEREY NAGEL 01 2012 Rev 02 31 PCR clean up gel extraction 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
22. ially primers are completely removed If you need to purify short ssDNA the additional Binding Buffer NTC can be used see Figure 8 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 1 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 2 Bind DNA Continue with step 2 of the protocol for PCR clean up section 5 1 490 bp 490 bases 164 bp 164 bases 100 bases 64 bases 18 bases u NTI NTC Figure 8 Purification of dsDNA and ssDNA using buffers NTI and NTC PCR fragments amplified using one phosphorylated and one dephosphorylated primer were partially digested with Exonuclease to yield single stranded DNA Samples were purified using Binding Buffer NTI 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 Buffer NTI removes ssDNA lt 150 bases NTI lane 2 whereas Buffer NTC leads to full recovery of even primer
23. ition by GuHCl light gray and GuSCN dark gray A 164 bp DNA fragment was amplified from 5 ng pBS template with DyNAmo Capillary Master Mix NEB in a Lighcycler real time PCR machine Roche in the presence of 0 80 mM GuHCl or GuSCN Salt carry over always happens with both GuSCN and GuHCl and could only be minimized by extensive washing This however is unnecessary since the final concentration of chaotropic salt in eluates is much too small to have any negative effect As a result a non ideal A A can simply be ignored 14 MACHEREY NAGEL 01 2012 Rev 02 PCR clean up gel extraction 3 Storage conditions and preparation of working solutions Attention Buffer NTI contains chaotropic salt Wear gloves and goggles CAUTION Buffer NTI contains guanidinium thiocyanate which can form highly reactive compounds when combined with bleach sodium hypochlorite DO NOT add bleach or acidic solutions directly to the sample preparation waste Storage conditions The NucleoSpin Gel and PCR Clean up kit should be stored at room temperature and is stable for at least one year Before starting any NucleoSpin Gel and PCR Clean up 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 Gel and PCR C
24. lean up 10 preps 50 preps 250 preps REF 740609 10 740609 50 740609 250 Wash Buffer NT3 6 mL 20 mL 2x50 mL Concentrate Add 24 mL ethanol Add 80 mL ethanol Add 200 mL ethanol to each bottle MACHEREY NAGEL 01 2012 Rev 02 15 PCR clean up gel extraction 4 Safety instructions The following components of the NucleoSpin Gel and PCR Clean up kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section 4 1 Risk and safety phrases Component Hazard contents Hazard Risk Safety symbol phrases phrases Inhalt Gefahrstoff Gefahrstoff R S tze S S tze symbol Guanidinium thiocyanate x Xn R 20 21 22 S 13 61 Guanidiniumthiocyanat 32 52 53 NTI Risk phrases R 20 21 22 R 32 R 52 53 Harmful by inhalation in contact with skin and if swallowed Gesundheitssch dlich beim Einatmen Verschlucken und Ber hrung mit der Haut Contact with acids liberates very toxic gas Entwickelt bei Ber hrung mit S ure sehr giftige Gase Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment Sch dlich f r Wasserorganismen kann in Gew ssern l ngerfristig sch dliche Wirkungen haben Safety phrases S13 R 61 Keep away from food drink and animal feedstuffs Von Nahrungsmitteln Getr nken und Futtermitteln fernhalten Avoid release to the environment Refer to special instructions safety data sheet Freisetzung in
25. ley es xg through which contains the DNA z 1 min Optional To increase the final yield repeat step 3 and 4 Keep flow and combine both supernatants or flow throughs eS through 22 MACHEREY NAGEL 01 2012 Rev 02 NucleoSpin Gel and PCR Clean up 5 Adjust DNA binding condition Mix 1 volume of sample with 2 volumes of Buffer NTI e g 200 uL diffusion buffer and 400 uL of Buffer NTI Small amounts of precipitating SDS do not influence the purification Do not remove the precipitate Note To obtain higher yields for small fragments lt 50 bp add two volumes of ethanol or use Buffer NTC instead of Buffer NTI Buffer NTC is not provided with the kit but can be ordered separately see ordering information Bind DNA Continue with step 2 of the protocol for PCR clean up section 5 1 2 vol NTI per 1 vol sample Optional 2vol ethanol or 2 vol NTC per 1 vol sample MACHEREY NAGEL 01 2012 Rev 02 23 NucleoSpin Gel and PCR Clean up 5 4 RNAextraction from agarose gels Buffer NTC Not only DNA but also RNA can be extracted from agarose gels To efficiently bind especially the small single stranded RNA Binding Buffer NTC has to be used instead of standard Binding Buffer NTI To fractionate RNA run a standard RNA gel with denaturing RNA loading buffer but do not use formaldehyde or glyoxal These compounds not only inactivate RNases and denature RNA but also modify RNA A
26. lt 6 0 Figure 2 If the pH increases to around 7 after adding the sample the solution will turn green In addition an even higher pH will be signaled by a blue color If a change in color is observed the pH should be corrected by adding more Buffer NTI or by titrating the pH back to lt 6 0 with 4 M sodium acetate pH 5 0 or small amounts of hydrochloric acid HCI 2 5 Tips and tricks for extractions from agarose gels Subject Recommendation Buffer system Running conditions Cutting out the band Size of gel piece TBE Tris Borate EDTA buffer has a higher buffering capacity than TAE Tris Acetate EDTA which is needed for runs overnight and offers a better resolution for small DNA fragments TBE buffer can be used in combination with NucleoSpin Gel and PCR Clean up However it is preferred to use fresh TAE buffer over TBE for preparative agarose gels TAE does not interact with agarose resulting in higher DNA yields Additionally linear DNA runs faster and the resolution of large DNA fragments is higher Furthermore supercoiled plasmid is separated better from linear and open circle DNA The temperature during electrophoresis should be low in order to increase the resolution of the DNA separation and avoid melting of the gel thus causing denaturation of the DNA Use fresh buffer and run the gel at low voltage lt 60 V for as short as possible As soon as the DNA band of interest is sufficiently separated from th
27. nts larger than 20 kbp may be mechanically damaged by the fast centrifugation through the membrane For very large fragments consider using NucleoTrap or NucleoTraP CR see ordering information To elute the DNA water with a pH gt 7 is needed to reestablish the hydrate shell It is highly recommended to elute DNA with Elution Buffer NE 5 mM Tris HCl pH 8 5 which is provided with the kit However a standard TE buffer may also be used to ensure best elution efficiency Please note that EDTA in TE buffer may cause problems in subsequent enzymatic reactions Do not use deionized water since its pH is usually too acidic If even less salt than the 5 mM Tris has to be used dilute Elution Buffer NE with distilled water and make sure the pH is still gt 7 Unbuffered elution buffer should not be used The standard elution buffer volume is 15 30 uL which is the best compromise for high DNA recovery and high DNA concentration for fragments lt 1000 bp Figure 3 100 80 60 40 A a atk 20 0 50 bp 400 bp 250 bp 500 bp 750 bp 1 kbp 3 kbp 5 kbp Fragment length DNA recovery Figure 3 Fragment length dependent DNA recovery 2 ug of 100 bp DNA ladder Fermentas or 2 ug of linearized vectors of 3 and 5 kbp were purified from standard PCR buffer or 200 mg 1 TAE agarose gel DNA was eluted in 30 uL Elution Buffer NE MACHEREY NAGEL 01 2012 Rev 02 11 PCR clean up gel extraction Elution after
28. posable pipette tips Equipment Manual pipettors Centrifuge for microcentrifuge tubes Heating block water bath or thermomixer for gel extraction Scalpel to cut agarose gels 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 user manual if the NucleoSpin Gel and PCR Clean up 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 Please contact Technical Service regarding information about changes of the current user manual compared to previous revisions MACHEREY NAGEL 01 2012 Rev 02 5 PCR clean up gel extraction 2 2 1 Product description The basic principle NucleoSpin Gel and PCR Clean up is developed as a 2 in 1 kit allowing DNA fragments to be purified from enzymatic reactions such as PCR as well as from agarose gels The sample is mixed with Binding Buffer NTI and in case of a cut out gel band it is heated to dissolve the agarose In the presence of chaotropic salt the DNA is bound to the silica membrane of a NucleoSpin Gel and PCR Clean up Column Contaminations are removed by simple washing steps with ethanolic Wash
29. purifying the 164 bp fragment with gt 90 8 MACHEREY NAGEL 01 2012 Rev 02 PCR clean up gel extraction 1 Pa gi Or BIO 12 ES zZ bp nt _ 982 645 359 a ca a as 164 100 79 65 50 21 VoINT 1 1 1 1 1 1 1 1 1 1 Volwater 0 1 2 3 4 5 6 7 8 9 NTI 100 50 33 25 20 17 14 13 11 10 Figure 1 Purification of PCR reactions using Buffer NTI 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 NTI Lane 4 12 Purification with Buffer NTI diluted with 1 9 volumes of water 2 4 pH indicator The optimal pH to bind even small DNA fragments to the silica membrane of the NucleoSpin Gel and PCR Clean up Columns is approximately 5 0 6 0 The Binding Buffer NTI is sufficiently buffered to maintain this pH for all standard PCR reaction buffers or agarose gel buffer systems In addition the colored binding buffer helps identify undissolved pieces of agarose during DNA gel extraction 12 104 pH Fann Do yellow Figure 2 Titration curve of Binding Buffer NTI with pH indicator MACHEREY NAGEL 01 2012 Rev 02 9 PCR clean up gel extraction A yellow color indicates the optimal pH
30. r 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 net com Weiterf hrende Informationen finden Sie in den Sicherheitsdatenbl ttern www mn net com MACHEREY NAGEL 01 2012 Rev 02 17 NucleoSpin Gel and PCR Clean up 5 Protocols 5 1 PCR clean up The following protocol is suitable for PCR clean up as well as DNA concentration and removal of salts enzymes etc from enzymatic reactions SDS lt 0 1 Before starting the preparation Check if Wash Buffer NT3 was prepared according to section 3 1 Adjust DNA binding condition For very small sample volumes lt 30 uL adjust the volume of the reaction mixture to 50 100 uL with water It is not necessary to remove mineral oil 2 vol NTI r Mix 1 volume of sample with 2 volumes of Buffer NTI Pa 1 vol sample e g mix 100 uL PCR reaction and 200 uL Buffer NTI Note For removal of small fragments like primer dimers dilutions of Buffer NTI can be used instead of 100 Buffer NTI Please refer to section 2 3 2 Bind DNA Place a NucleoSpin Gel and PCR Clean up Column into a Collection Tube 2mL and load up to 700 uL Load sample sample Centrifuge for 30s at 11 000 x g Discard flow through and place the column back into the collection tube 1 pong xg s Load remaining sample if necessa
31. rom agarose gels section 5 2 24 MACHEREY NAGEL 01 2012 Rev 02 NucleoSpin Gel and PCR Clean up 5 5 DNA clean up of samples containing SDS Buffer NTB Buffer NTI from the NucleoSpin Gel and PCR Clean up kit is compatible with most commonly used detergents with the exception 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 1 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 uL 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 PCR clean up section 5 1 MACHEREY NAGEL 01 2012 Rev 02 25 NucleoSpin Gel and PCR Clean up 5 6 Single stranded DNA clean up Buffer NTC Buffer NTI from the NucleoSpin Gel and PCR Clean up kit is able to bind single stranded DNA ssDNA gt 150 bases Shorter oligonucleotides espec
32. ry and repeat the centrifugation step 3 Wash silica membrane 700 uL NT3 Add 700 uL Buffer NT3 to the NucleoSpin Gel and PCR H Clean up Column Centrifuge for 30 s at 11 000 x g Discard flow through and place the column back into the 11 000 x g collection tube 30s 18 MACHEREY NAGEL 01 2012 Rev 02 NucleoSpin Gel and PCR Clean up Recommended Repeat previous washing step to minimize chaotropic salt carry over and improve A A values see section 2 7 for detailed information 700 HENTI 11 000 x g 30s Dry silica membrane Centrifuge for 1 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 BufferNT3 might inhibit ES 11 000 x g enzymatic reactions Total removal of ethanol can be 1 min achieved by incubating the columns for 2 5 min at 70 C prior to elution Elute DNA Place the NucleoSpin Gel and PCR Clean up Column 15 30 pL NE into a new 1 5 mL microcentrifuge tube not provided Add 15 30 uL Buffer NE and incubate at room RT temperature 18 25 C for 1 min Centrifuge for 1 min 1 min at 11 000 x g Note DNA recovery of larger fragments gt 1000 bp can be cs u va eg increased by multiple elution steps with fresh buffer heating min to 70 C and incubation for 5 min See section 2 6 for detailed information
33. s a result the RNA yield is significantly reduced and more important the RNA may not work properly in enzymatic downstream applications such as RT PCR or in vitro transcriptions Without formaldehyde the RNA is very sensitive to contaminating RNases Use gloves and make sure all equipment is RNase free especially the agarose and the running buffers Run the gel as short and as cold low voltage cold room as possible Note that the RNA may form secondary structures and may run differently from denaturing agarose gels 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 1 Excise RNA fragment solubilize gel slice Note Minimize UV exposure time to avoid damaging the RNA Refer to section 2 5 for more tips on agarose gel extraction Take a clean scalpel to excise the RNA fragment from an agarose gel Remove all excess agarose Determine the weight of the gel slice and transfer it to a e clean tube For each 100 mg of agarose gel lt 2 add 200 uL 200 pL NTC Buffer NTC per For gels containing gt 2 agarose double the volume of 100 mg gel Buffer NTC Incubate sample for 5 10 min at 50 C Vortex the sample 50 C briefly every 2 3 min until the gel slice is completely i 5 10 min dissolved 2 Bind RNA Continue with step 2 of the protocol for DNA extraction f
34. urity ratio A A if DNA is contaminated with chaotropic salts Carry over of GuSCN can lower the ratio from its ideal value of gt 2 0 to below 1 5 or even 1 0 GuHCl on the other hand is invisible at this wave length and does not alter the ratio at all This effect MACHEREY NAGEL 01 2012 Rev 02 13 PCR clean up gel extraction however is only detectable with very small amounts of DNA such as typical yields of PCR reactions or gel extractions Technical advances in UV VIS spectrometry now allows measuring these small amounts of DNA in small volumes thus raising concerns that the DNA might be too dirty However this problem does not usually occur with larger amounts of DNA since its own absorption at 230 nm masks small contributions of any contamination The concentration of contaminating chaotropic salt is usually in the range of 100 uM to 1 mM and does not have any negative influence on enzymatic downstream applications for example PCR restriction or ligation Figure 7 shows qPCR inhibition by GUSCN and GuHCl demonstrating that PCR only starts to be inhibited by chaotropic salts with approximately a100 fold higher concentration 40 mM In addition it distinctly shows that not only can GuSCN cause inhibition but also that photometrically invisible GuHCI can as well 30 GuHCl 25 E GuSCN 20 4 104 i tn a 0 i i OuM 100uM 1mM 10mM 20mM 40 mM 60 mM 80 mM Cp value a Figure 7 qPCR inhib
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