NucleoSpin Extraction Kits User Manual

NucleoSpin Extraction Kits User Manual See List of Components for storage conditions FOR RESEARCH USE ONLY PT3631-1 (PR24221) Published 22 April 2002

Table of Contents I. Introduction 3 II. List of Components 5 III. Additional Materials Required 6 IV. NucleoSpin Extract Protocols 7 A. Extraction from Agarose Gels 7 B. Isolation from PCR 8 V. NucleoSpin 8 Extract Purification Protocol 10 VI. NucleoSpin 96 Extract Purification Protocol 13 VII. Troubleshooting 16 VIII. Related Products 18 List of Figures Figure 1. NucleoSpin Multi-8 and NucleoVac manifold components. 3 Figure 2. NucleoVac-96 manifold and accessory components. 4 Figure 3. The NucleoSwing Z 513 Benchtop Centrifuge. 4 Figure 4. Assembling the manifold before the wash steps. 11 Figure 5. The fully assembled manifold. 12 Figure 6. Reassembling the manifold for elution into Collection Tubes. 12 Figure 7. Assembling the NucleoVac-96 manifold. 14 Notice to Purchaser This product is intended to be used for research purposes only. It is not to be used for drug or diagnostic purposes, nor is it intended for human use. Clontech products may not be resold, modified for resale, or used to manufacture commercial products without written approval of Clontech Laboratories, Inc. covered by patents,including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration, is hereby granted by implication or estoppel. Further information on purchasing licenses to practice the PCR Process where the process is covered by patents may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California94404 or the Licensing Department at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California 94501 NucleoSpin and NucleoBond are registered trademarks of MACHEREY-NAGEL GmbH & Co. KG. NucleoVac TM and NucleoSwing TM are trademarks of Macherey-Nagel GmbH and Co. Clontech, Clontech logo and all other trademarks are the property of Clontech Laboratories, Inc. Clontech is a Takara Bio Company. 2005 Clontech Laboratories, Inc. www.clontech.com Protocol # PT3631-1 Version # PR24221

I. Introduction NucleoSpin Kits NucleoSpin Kits provide a fast, efficient, and economical means of purifying nucleic acids from small volumes of bacterial or yeast cultures, PCR products, agarose gels, blood, tissues, viruses, or plants. These kits are designed around a unique polypropylene spin column manufactured by Macherey-Nagel GmbH and Co. Each spin column contains a specially activated silica membrane filter that traps nucleic acids and separates them from contaminating proteins and other cellular debris. After contaminants are spun through the filter, nucleic acids are eluted in a low-salt buffer (DNA) or RNase-free water (RNA). This manual provides methods for isolating DNA from agarose gels or from PCR reactions using the NucleoSpin Extract Kits. Samples in NucleoSpin columns can be processed either by centrifugation or, for more convenient processing of multiple samples simultaneously, by using vacuum filtration. All NucleoSpin columns contain a Luer-lock fitting that is compatible with the NucleoVac Vacuum manifold as well as several other vacuum manifolds. Additional 2-ml collection tubes (without lids) can be ordered separately. NucleoSpin Kits are available for a wide range of applications. See Related Products for ordering information. NucleoSpin High-Throughput Kits The NucleoSpin High-Throughput Kits provide uncompromised performance and convenience for purifying nucleic acids. Up to 96 samples can be processed simultaneously in under 90 minutes, without hazardous and time-consuming phenol/chloroform extractions or alcohol precipitations. Purified DNA can be used for fluorescent or radioactive sequencing, PCR, library screening, and cloning. This manual provides methods for the NucleoSpin 8 Extract Kits and the NucleoSpin 96 Extract Kits. The NucleoSpin 8 Extract Kits must be used with the NucleoVac TM vacuum manifold (Figure 1), which provides high-throughput purification capability through vacuum filtration in a 96-well format. Furthermore, the manifold delivers consistent and reproducible results from batch to batch. The multi-8 columns, attached to each other in strips of eight, contain a unique silica membrane that binds DNA in the presence of chaotropic salt. Filtrates are drawn out of the columns by vacuum, eliminating the need for centrifugation during the washing and eluting steps. This process significantly reduces prep time. The purified DNA is eluted into special Collection Tubes, which can be capped and stored for later use. The manifold and the NucleoSpin Multi-8 Kits are sold separately. Rack with Tube Strips column holders manifold base manifold lid Collection Tube Strips Cap Strips spacers Multi- Plasmid Strips wash plate Multi- Filter Strips waste reservoir Figure 1. NucleoSpin Multi-8 and NucleoVac TM manifold components. Protocol # PT3631-1 www.clontech.com Clontech Laboratories, Inc. Version # PR24221 3

I. Introduction The NucleoSpin Multi-96 Plates have a plate designed for easy handling and processing. The NucleoSpin 96 Extract Plates are compatible with the NucleoVac-96 vacuum manifold (Figure 2) making it easy to purify DNA from plasmid or PCR mixtures. These plates can also be used with the NucleoSwing TM Z 513 high-speed Benchtop Centrifuge (#4072-1; Figure 3) or an equivalent microtiter centrifuge; purified DNA is eluted into a Round-Well Block during centrifugation. The NucleoSwing Z 513 Benchtop Centrifuge (Figure 3) was specifically developed by Hermle (Wehingen, Germany) and Macherey-Nagel for high-throughput purification of nucleic acids using NucleoSpin Multi-96 Plates. In addition, a wide variety of fixed-angle and swinging bucket rotors, provided by Hermle, make this centrifuge ideal for all laboratory purposes. All parameters such as speed, RCF value, operating time, radius correction, and brake intensity are shown on the digital display. NucleoSwing and NucleoSpin 96 Kits are sold separately. DEEP - WELL BLOCK DEEP - WELL BLOCK SQUARE - WELL BLOCK SQUARE - WELL BLOCK MICROTUBE RACK MICROTUBE RACK Figure 2. NucleoVac-96 manifold and accessory components. Figure 3. The NucleoSwing TM Z 513 Benchtop Centrifuge. Clontech Laboratories, Inc. www.clontech.com Protocol # PT3631-1 4 Version # PR24221

II. List of Components Store all components at room temperature. NucleoSpin Extract Kits 635961 250 NucleoSpin Extract Columns (yellow) 250 2-ml Collection Tubes 3 x 100 ml Buffer NT1 150 ml Buffer NT2 (wash buffer) 80 ml Buffer NT3 (wash buffer) 50 ml Buffer NE (elution buffer) NucleoSpin 8 Extract Kit 635977 635978 50 ml 250 ml Buffer NT2 60 ml 300 ml Buffer NTW (wash buffer) 2 x 20 ml 200 ml Buffer NT3 (concentrated wash buffer) 20 ml 100 ml Buffer NE (elution buffer) 12 60 NucleoSpin 8 Extract Strips (yellow columns) 12 60 Collection Tube Strips and Rack 15 75 Cap Strips 1 5 Wash Plate 4 20 Self-Adhesive Aluminum Foils 4 20 Filter Paper, 125 mm x 85 mm NucleoSpin 96 Extract Kit 635993 635995 240 ml 75 ml Buffer NTB 200 ml 2 x 20 ml Buffer NT3 (concentrated wash buffer) 125 ml 25 ml Buffer NE (elution buffer) 4 1 NucleoSpin 96 Extract plates (yellow columns) 4 1 Round-Well Plates 4 1 Wash Plates (with adhesive foil) 4 1 Elution Plates (U-bottom) 8 2 Self-Adhesive Aluminum Foils Protocol # PT3631-1 www.clontech.com Clontech Laboratories, Inc. Version # PR24221

III. Additional Materials Required All NucleoSpin Kits 96 100% ethanol [Optional]: Matrix Impact2 electronic multichannel pipettor (see Related Products for ordering information.) 1.5-ml microcentrifuge tubes NucleoSpin Multi-8 Kits [Optional] Matrix round-well blocks for growing overnight cultures (see Related Products for ordering information.) 1.5-ml microcentrifuge tubes NucleoSpin 96 Extract Kits Collection tubes (in strips of eight) Clontech Laboratories, Inc. www.clontech.com Protocol # PT3631-1 6 Version # PR24221

IV. NucleoSpin Extract Protocols NucleoSpin Extract Kits are designed for extracting and isolating DNA fragments larger than 100 bp from agarose gels and PCR products. Average yields of 5 15 µg of DNA can be isolated per preparation. General considerations: Add the appropriate volume of 96-100% ethanol to Buffer NT3 before use. The appropriate volume is also printed on the Buffer NT3 bottle. The recommended elution buffer, Buffer NE, is 5 mm Tris-HCl (ph 8.5) and is included in the NucleoSpin Extraction Kit. You may also use TE buffer (ph 8.5) or sterile deionized H 2 O (ph 8.5). If you are using H 2 O, check the ph before use, because dissolved CO 2 from the air lowers the ph. Lower recoveries may result if the elution buffer becomes too acidic. If you are isolating 5 10 kb fragments, we recommend warming the Buffer NE to 70 C before use. After adding Buffer NE to the spin column, incubate the samples at room temperature for one minute to collect the purified fragment before centrifuging. Recovery rates for DNA fragments between 100 bp and 10 kb are typically 75 90%. Recovery rates for fragments smaller than 100 bp drop precipitously; we recommend using the NucleoTrap Gel Extraction Kit to isolate small fragments. See Related Products for ordering information. DNA fragments can be isolated from TAE or TBE gels made from either normal or low-melting-point agarose. TAE and TBE gels are normally ph 6 7.5, which is the optimal ph for adsorption of DNA fragments onto the NucleoSpin membrane. If in doubt, check the ph of the gel running buffer before adding the agarose gel slice into the NucleoSpin column. A. Extraction from Agarose Gels 1. Use a clean scalpel or razor blade to excise the DNA fragment from the agarose gel. Excise the fragment carefully to minimize the volume of agarose loaded onto the NucleoSpin membrane. Weigh the gel slice, and then transfer it to a clean microcentrifuge tube or 15-ml conical tube. 2. For every 100 mg of agarose, add 300 µl of Buffer NT1. Double the volume of NT1 for gels made with >2% agarose. 3. Incubate the sample at 50 C for 10 min; briefly vortex the sample every 2 3 min during incubation. 4. Insert the NucleoSpin Extract Spin Column into a 2-ml collection tube. Load the sample into the column. Centrifuge at 8,000 x g for 1 min at room temperature. Discard the flowthrough. mix sample + Buffer NT1 10 min, 50 C Load sample 1 min, 8,000 x g 5. Reinsert the column into the 2-ml Collection Tube. Add 600 µl of Buffer NT3 to the column. Centrifuge at 11,000 x g for 1 min. Discard the flowthrough. Add 600 µl Buffer NT3 1 min, 11,000 x g Protocol # PT3631-1 www.clontech.com Clontech Laboratories, Inc. Version # PR24221

IV. NucleoSpin Extract Protocols continued 6. Add 200 µl of Buffer NT3 to the column. Centrifuge at 11,000 x g for 2 min. Note: The ethanol in Buffer NT3 inhibits enzymatic reactions and must be removed completely by performing this centrifugation step. Add 200 µl Buffer NT3 2 min @ 11,000 x g 7. Place the NucleoSpin Extract column in a clean 1.5-ml microcentrifuge tube. Add 25 50 µl of Buffer NE and incubate 1 min to increase the yield of eluted DNA. Centrifuge at 11,000 x g for 1 min. Note: For higher yields, we recommend eluting twice with 25 µl each time. Yields of larger DNA fragments (>5 10 kb) can be increased by using prewarmed elution buffer (70 C). 8. Determine yields by UV spectrophotometry and by agarose gel electrophoresis. Add 25 50 µl Buffer NE 1 min @ 11,000 x g B. Isolation from PCR Reactions 1. After PCR is complete, remove tube(s) from the thermal cycler. It is not necessary to remove the mineral oil layer from the PCR sample. However, if necessary, the oil layer can be removed by placing tubes at 20 C for about 30 min to freeze the aqueous layer. Remove the oil layer using a pipette. 2. Adjust the reaction volume to 100 µl with TE (ph 7 7.5). 3. Add 400 µl of Buffer NT2 to the sample. Mix well. mix sample Add 400 µl Buffer NT2 4. Insert the NucleoSpin Extract Spin Column into a 2-ml collection tube. Load the sample into the column. Centrifuge at 11,000 x g for 1 min at room temperature. Discard the flowthrough. Load sample 1 min @ 11,000 x g 5. Reinsert the column in the 2-ml Collection Tube. Add 600 µl of Buffer NT3 to the column. Centrifuge at 11,000 x g for 1 min at room temperature. Discard the flowthrough. Add 600 µl Buffer NT3 1 min @ 11,000 x g Clontech Laboratories, Inc. www.clontech.com Protocol # PT3631-1 Version # PR24221

IV. NucleoSpin Extract Protocols continued 6. Put the NucleoSpin Extract column back in the 2-ml Collection Tube and add 200 µl Buffer NT3. Centrifuge at 11,000 x g for 2 min to remove residual Buffer NT3 from the membrane filter. Note: The ethanol in Buffer NT3 inhibits enzymatic reactions and must be removed completely by performing this centrifugation step. Add 200 µl Buffer NT3 2 min @ 11,000 x g 7. Place the NucleoSpin Extract column in a clean 1.5-ml microcentrifuge tube. Add 25 50 µl of Buffer NE and incubate at room temperature for 1 min. Centrifuge at 11,000 x g for 1 min. Note: Yields of larger DNA fragments (>5 10 kb) can be increased by using prewarmed elution buffer (70 C). Add 25 50 µl Buffer NE 1 min @ 11,000 x g 8. Determine yields by UV spectrophotometry and by agarose gel electrophoresis. Protocol # PT3631-1 www.clontech.com Clontech Laboratories, Inc. Version # PR24221

V. NucleoSpin 8 Extract Purification Protocol This kit is designed to purify PCR products from reaction mixtures. The purified DNA is of high quality and can be used directly for sequencing. Recovery rates of 75 90% can be achieved for fragments between 100 bp to 10 kb. Primers, nucleotides, salts, and polymerases are effectively removed using these columns; up to 96 samples can be processed simultaneously in less than 60 minutes. A. General Considerations The specially treated silica membrane allows purification of up to 15 µg of DNA. The adsorption of DNA to the NucleoSpin PCR membrane is ph dependent. High yields are achieved at ph<8. The elution efficiency depends on the ph of the elution buffer. Elution is most effective at a ph 7.5 8.5. When using Buffer NE (5 mm Tris-HCl, ph 8.5) or deionized water, check and adjust the ph to 8.5; lower ph may lead to low recoveries. For increased yields of large DNA fragments (>5 10 kb), prewarm the elution buffer to 70 C. We recommend using an elution volume of 75 125 µl NE Buffer (included with the kit), as well as a 3 5 min incubation of the elution buffer on the membrane. B. Before you start Add 80 ml of 96 100% ethanol to each bottle of Buffer A4 before use. Establish a reliable vacuum source for the NucleoVac. The manifold may be used with a water aspirator, house vacuum, or a vacuum pump. We recommend a vacuum of 200 400 mbar (2.9 5.8 psi). As an alternative to measuring the exact pressure, adjust the vacuum so that the PCR solution flows through the column at a rate of 1 2 drops per second during purification. C. Procedure for Purifying PCR Products 1. The removal of mineral oil is not necessary. However, if desired, it can be removed without chloroform extraction in the following manner: freeze the aqueous phase by storing the PCR samples for 30 min at 20 C. Remove and discard the organic phase by pipetting. 2. Add four volumes of NT2 Buffer to one volume of the PCR mix (e.g., for 100 µl of reaction mix, add 400 µl NT2 Buffer). Mix well. For multiple PCR samples, transfer the reaction mix to a suitable deep-well microtiter plate, pre-filled with appropriate amount of NT2 Buffer. Mix well. 3. Prepare the vacuum manifold. Figure 3 illustrates the manifold setup: a. Insert the clear plastic spacers labeled D, notched side up, into the grooves located on the short sides of the manifold. Place the waste reservoir inside the manifold base. b. Affix the adhesive foil onto the top of the bottomless wash plate and rest the wash plate on the spacers. c. Insert the appropriate number (up to 12) of NucleoSpin 8 Extract Strips (yellow) into the openings of the column holder. Make sure the column holder is right side up. The columns should fit snugly. Seal any unused openings of the column holder with Cap Strips. d. Insert the column holder into the lid of the manifold. e. Place the lid firmly on top of the manifold base to create a tight seal. Press the column holder down in order to pierce the foil on top of the wash plate. Clontech Laboratories, Inc. www.clontech.com Protocol # PT3631-1 10 Version # PR24221

V. NucleoSpin 8 Extract Purification Protocol continued NucleoSpin Extraction Kits User Manual column holder manifold lid wash plate with foil attached "D" "D" waste reservoir (open) Figure 4. Assembling the NucleoVac manifold before the wash steps. 4. Load the samples from Step IV.C.2 onto the yellow columns already placed on the manifold. Apply vacuum by slowly opening the valve. Press down on the column holder to establish a good seal, and allow the buffer to flow through the columns. Adjust vacuum to establish a flow rate of 1 2 drops per second. Important note: The majority of laboratory vacuum setups, when set to the maximum, produce a vacuum that exceeds the recommended pressure for the manifold. To achieve acceptable yields, adjust the strength of the vacuum before you elute the plasmid DNA. Adjust the vacuum a this step, or the following wash steps, to achieve the recommended flow rate. 5. After samples are filtered through, vent the manifold by closing the valve. 6. Add 500 µl of Buffer NTW to each column. This step is recommended for removal of primers longer than 40 nucleotides and for primer dimers. 7. Open the valve to apply vacuum. Again, press down on the column holder and allow all the buffer to drain out of the columns. Close the manifold valve. 8. Add 900 µl of Buffer NT3 to each column. Open the valve to apply vacuum. After all the buffer has drained out of the columns, close the valve again. 9. Repeat Step 8, and vacuum for 5 min. 10. Keeping the valve closed, remove the column holder from the manifold. Remove any residual wash buffer from the columns and waste reservoir and, if necessary, blot the tips of the columns with filter paper to dry. Remove the wash plate and spacers from inside the manifold, and reinsert the column holder with the columns intact onto the manifold lid. 11. Open the valve to apply vacuum for 10 min to dry the membranes completely. This step is extremely important; traces of ethanol left on the filters may interfere with subsequent enzymatic reactions. 12. After the filters are completely dry, close the valve to vent the manifold. Protocol # PT3631-1 www.clontech.com Clontech Laboratories, Inc. Version # PR24221 11

V. NucleoSpin 8 Extract Purification Protocol continued Figure 5. The fully assembled manifold. 13. Elute the DNA into Collection Tubes (provided with the kit) or into a microtiter plate (not provided): a. To elute into Collection Tubes, insert spacers labeled "F" into the grooves in the short sides of the manifold. Rest the collection tube rack, containing the appropriate number of Collection Tubes, onto the spacers. Reassemble the manifold as shown in Figure 5. b. To elute into a microtiter plate, insert the spacers labeled "D" into the grooves, place a standard microtiter plate onto the spacers, and reassemble the manifold. 14. Add 75 125 µl of Buffer NE (elution buffer) or H 2 O (ph 7.5 8.5) to the columns. Pipet each aliquot of buffer directly onto the center of the membrane to ensure rapid and complete dissolution of the DNA into the buffer. Allow the buffer to remain on the membrane for 1 5 min. 15. Open the valve to apply the vacuum and collect the eluted DNA. Close the valve to vent the manifold. The eluted DNA is now ready for further use. Note: The "dead volume" of each column is about 50 µl. You should be able to collect 85 100 µl of the eluate in each Collection Tube. column holder manifold lid Collection Tubes & Rack "F" "F" Figure 6. Reassembling the manifold for elution into Collection Tubes. If you are eluting into a microtiter plate, substitute the plate for the Collection Tubes in the same position. Use plates labeled "D" for microtiter plate. Clontech Laboratories, Inc. www.clontech.com Protocol # PT3631-1 12 Version # PR24221

VI. NucleoSpin 96 Extract Purification Protocol NucleoSpin Extraction Kits User Manual This kit is designed to purify PCR products from reaction mixtures. The purified DNA is of high quality and can be used directly for sequencing. Recovery rates of 75 90% can be achieved for fragments between 100 bp to 10 kb. Primers, nucleotides, salts, and polymerases are effectively removed using these columns; up to 96 samples can be processed simultaneously in less than 45 minutes. A. General Considerations The specially treated silica membrane allows purification of up to 15 µg of DNA. The adsorption of DNA to the NucleoSpin PCR membrane is ph dependent. High yields are achieved at ph<8. The elution efficiency depends on the ph of the elution buffer. Elution is most effective at a ph 7.5 8.5. When using Buffer NE (5 mm Tris-HCl, ph 8.5) or deionized water, check and adjust the ph to 8.5; lower ph may lead to low recoveries. For increased yields of large DNA fragments (>5 10 kb), prewarm the elution buffer to 70 C. We recommend using an elution volume of 75 125 µl NE Buffer (included with the kit), as well as a 3 5 min incubation of the elution buffer on the membrane. B. Before you start Add 95% ethanol to each bottle of Buffer NT3 before use. The amount of volume is indicated on the Buffer NT3 bottle. For PCR mixture volumes less than 100 µl, add Tris buffer (10 mm, ph 7) and adjust the volume to 100 µl. Establish a reliable vacuum source for the NucleoVac. The manifold may be used with a water aspirator, house vacuum, or a vacuum pump. We recommend a vacuum of 200 400 mbar (2.9 5.8 psi). As an alternative to measuring the exact pressure, adjust the vacuum so that during the purification procedure, the PCR solution flows through the column at a rate of 1 2 drops per second. C. Procedure for Purifying PCR Products 1. The removal of mineral oil from the reaction tube is not essential However, if necessary, oil can be removed without chloroform extraction by freezing the aqueous phase by storing the PCR samples for 30 min at 20 C. Remove and discard the mineral oil by pipetting. 2. Add five volumes of NTB Buffer to one volume of the PCR mix (e.g., for 100 µl of reaction mix, add 400 µl NT2 Buffer). Mix well. For multiple PCR samples, transfer the reaction mix to a suitable Round-Well Plate (provided), pre-filled with appropriate amount of NTB Buffer. Mix well. 3. Prepare the vacuum manifold. Figure 7 illustrates the manifold setup: a. Insert the clear plastic spacers labeled MTP/Multi-96 Plate, notched side up, into the grooves located on the short sides of the manifold. b. Insert the wash plate, with the foil on its top side, on the spacers (left). c. Insert the NucleoSpin Multi-96 Extract plate (yellow) into the manifold s lid. d. Place the lid firmly on top of the manifold base to create a tight seal, and press down on the plate in order to pierce the foil on top of the wash plate. Close the vacuum manifold. 4. Load the samples from Step V.C.2 into the 96 Extract plate, which is placed on the manifold. Close unused openings with self-adhesive foil (provided). Apply vacuum by slowly opening the valve. Press down on the column holder to establish a good seal, and allow the buffer to flow through the columns. Adjust vacuum to establish a flow rate of 1 2 drops per second. Important note: The majority of laboratory vacuum setups, when set to the maximum, produce a vacuum that exceeds the recommended pressure for the manifold. To achieve acceptable yields, adjust the strength of the vacuum before you elute the plasmid DNA. Adjust the vacuum at this step, or the following wash steps, to achieve the recommended flow rate. 5. After samples are filtered through, vent the manifold by closing the valve. Protocol # PT3631-1 www.clontech.com Clontech Laboratories, Inc. Version # PR24221 13

VI. NucleoSpin 96 Extract Purification Protocol continued Figure 7. Assembling the NucleoVac-96 manifold. 6. [Optional]: Add 500 µl of Buffer NT3 to each column. Open the valve to apply vacuum. Again, press down on the plate and allow all the buffer to drain out of the columns. Close the manifold valve. Note: This step is recommended for removing primers >40 nucleotides and for primer dimers. We strongly recommend this wash step if you plan to sequence your purified products. 7. Add 900 µl of Buffer NT3 to each well. Open the valve to apply vacuum. After all the buffer has drained out of the plate, close the valve again. 8. Repeat Step 7, and apply vacuum for 5 min. D. Elution of DNA using the Vacuum Manifold 1. Keeping the valve closed, remove the 96 Extract plate from the manifold. Remove any residual wash buffer from the columns and waste reservoir and, if necessary, blot the tips of the columns with filter paper. Remove the wash plate, spacers, and waste reservoir from inside the manifold, and reinsert the manifold lid and 96 Extract plate. 2. Open the valve to apply vacuum for at least 10 min to dry the membranes completely. This step is extremely important; traces of ethanol left on the filters may interfere with subsequent enzymatic reactions. 3. After the filters are completely dry, close the valve to vent the manifold. Clontech Laboratories, Inc. www.clontech.com Protocol # PT3631-1 14 Version # PR24221

VI. NucleoSpin 96 Extract Purification Protocol continued NucleoSpin Extraction Kits User Manual 4. Elute the plasmid DNA into the Elution Plate (U-bottom; provided with the kit) or Collection Tubes (not provided). Assemble the NucleoVac-96 Manifold as shown in Figure 6: a. To elute into a Elution Plate, insert the spacers labeled MTP/Multi-96 Plate into the grooves, place the Elution Plate U-bottom (right) onto the spacers, and close by putting the manifold lid containing the 96 Extract Filter Plate on top. b. To elute into Collection Tubes, insert spacers labeled Microtube rack into the grooves in the short sides of the manifold. Rest the collection tube rack, containing the appropriate number of Collection Tubes, onto the spacers. 5. Add 100 125 µl of Buffer NE (elution buffer) or H 2 O (ph 7.5 8.5) to the columns. Pipet each aliquot of buffer directly onto the center of the membrane to ensure rapid and complete dissolution of the DNA into the buffer. Allow the buffer to remain on the membrane for 1 5 min. 6. Open the valve to apply the vacuum and collect the eluted DNA. Close the valve to vent the manifold. The eluted DNA is now ready for use. Note: The dead volume of each column is about 50 µl. You should be able to collect 85 100 µl of the eluate in each well. E. Elution of DNA using a Centrifuge Elution of purified DNA in a centrifuge may be necessary when higher final concentrations of the DNA are required for downstream applications. With a centrifuge, DNA can be eluted in 50 75 µl total volume resulting in concentrations of 70 200 µg/ml. 1. Cover the NucleoSpin Plasmid Plate with self-adhesive tape. Place the plate on top of the Round-well block and centrifuge for 10 minutes at maximum speed (5,800 x g optimal) to dry the membranes and the outlets of the binding plate. Note: We recommend Hermle/Macherey-Nagel: NucleoSwing Z513 or equivalent with a swing-out rotor capable of accommodating the NucleoSpin Plasmid Plate. Do not use a microtiter plate as a support for the NucleoSpin Extract Plate because microtiter plates may crack when centrifuged at > 2,500 x g. 2. Insert the Plasmid Plate into a new round-well block. Remove the self-adhesive tape and dispense 50 75 µl Buffer NE (elution buffer) directly onto the silica membrane. Incubate 1 3 min. 3. Centrifuge for 2 min at maximum speed (5,800 x g optimal) to collect the DNA. Remove the Round-well block containing the eluted DNA and seal with self-adhesive tape for storage. Protocol # PT3631-1 www.clontech.com Clontech Laboratories, Inc. Version # PR24221 15

VII. Troubleshooting A. Troubleshooting Tips for NucleoSpin Extract Protocols 1. Incomplete dissolution of gel slices High concentration of agarose Use a double volume of Buffer NT1 for high concentration agarose gels. Wrong buffer Buffer NT2 cannot be used for gel dissolution Time and temperature Depending on the weight and size of the gel slice, the incubation can be increased up to 20 minutes. Vortex every 2 minutes and check. 2. Low DNA yield Reagents not prepared properly Add 96 100 % ethanol to buffer NT3 and mix well before use. Insufficient drying of NucleoSpin Membrane Centrifuge at 11,000 x g for 5 min before elution to remove all traces of ethanol-containing buffer. Isolation of large DNA fragments Preheat elution Buffer NE to 70 C before applying to silica membrane. Incubate for 2 min before centrifuging. 3. Suboptimal performance in downstream applications Carry-over of ethanol-containing buffers Remove all ethanol-containing Buffer NT3 by centrifuging at 11,000 x g for 3-5 minutes. Elution of DNA buffers other than Buffer NE EDTA in TE buffer could inhibit sequencing reactions. If TE was used to elute, repurify the DNA and elute in Buffer NE or water. B. Troubleshooting Tips using the NucleoVac manifold and NucleoVac-96 manifold. Insufficient vacuum: Check the insertion of the 8 Extract Strips or 96 plate. Insert the NucleoSpin 8 Extract strips tightly and upright into the column holder by pressing down on the 8-well strips using one thumb on each side of the column holder. Be sure to press both sides of the 8-well strip. Strips placed unevenly will reduce the vacuum. Check the column holder. The column holder must be flat and straight. Place the column holder on a lab bench, and press down on its edges. Finally, close all openings of the column holder with the Cap Strips. Insert the holder into the cover lid, and close the vacuum manifold with the lid. Apply and test the vacuum. Check the O-rings. Apply the vacuum to the manifold. Then insert a microtiter plate with the rectangular bottom instead of the column holder. The O-ring of the vacuum manifold must be covered completely by the microtiter plate. Apply and test the vacuum. The microtiter plate should be held in place by the vacuum. Check the vacuum manifold. Are there any cracks on the surface of the vacuum manifold? Check the main valve. Is it tightly fixed into the vacuum manifold? Are there any cracks around the valve? Clontech Laboratories, Inc. www.clontech.com Protocol # PT3631-1 16 Version # PR24221

VII. Troubleshooting continued C. Troubleshooting for NucleoSpin 96 Extract Kit 1. Poor DNA yield Prepare Buffer NT3 before use Add the indicated volume of 95% ethanol to Buffer NT3 Check Elution Buffer Elution is most effective at a ph between 7.5 and 8.5. If you used water, make sure the ph is within this range. Always use a low salt buffer, such as Buffer NE or water, for elution. Check the elution volume The optimal elution buffer is 100 125 µl. We do not recommend using less than 75 µl. 2. Suboptimal performance of DNA in sequencing reactions Ethanol carry-over Be sure to remove Buffer NT3 after the final wash step. Dry the columns for at least 10 min. Elution of plasmid DNA with TE buffer EDTA may inhibit sequencing reactions; therefore, we do not recommend eluting with TE Buffer. Repurify DNA and elute using Buffer NE. Eluted DNA contains residual primers/primer dimers Minimize the amount of primers used in your PCR mixtures. Make sure to use a ratio of four to one: Buffer NT2 to PCR mixture. DNA concentration too low Quantitate DNA by Agarose gel electrophoresis before setting up sequencing reactions. Protocol # PT3631-1 www.clontech.com Clontech Laboratories, Inc. Version # PR24221 17

VIII. Related Products Product NucleoBond Plasmid Kits For the latest and most complete listing of all Clontech products, please visit www.clontech.com. Cat. No. Mini Kits 635925 635926 Midi Kits 635929 635930 635931 Maxi Kits 635933 635934 635935 Mega Kit 635938 Giga Kit 635939 NucleoBond BAC Maxi Kit 635941 NucleoSpin Plasmid Kit 635988 NucleoSpin Plasmid EF Kits Mega Kit 636036 Giga Kit 635955 NucleoSpin RNA II Kit 635990 635991 635992 NucleoSpin RNA L 636048 NucleoSpin Virus Kit Mini Kits 635970 635971 Midi Kit 635982 NucleoSpin Blood Kit Mini Kits 635963 635964 Midi Kit 635969 NucleoSpin Blood XL Kit 636049 636050 NucleoSpin Tissue Kit 635966 635967 NucleoSpin Plant Kit 635979 635980 NucleoSpin Food Kit 636044 636045 636046 NucleoSpin RNase A (100 mg) 635918 NucleoSpin T1 Lysis Buffer ( 25 ml) 635956 NucleoSpin High-Throughput Kits many Clontech Laboratories, Inc. www.clontech.com Protocol # PT3631-1 18 Version # PR24221

VIII. Related Products continued Product Cat. No. NucleoTrap Gel Extraction Kit 636018 NucleoTrap PCR Purification Kit 636020 NucleoSpin Collection Tubes (2-ml; 1,000 tubes) 636029 NucleoSpin Buffer Set 635957 NucleoSpin Buffer RA1 636037 The following products are available from Matrix Technologies Corporation. For more information, contact them at 800-345-0206 or visit their web site at www.matrixtechcorp.com: Round-Well block 8800-96S Matrix Impact2 electronic multichannel pipettor 2004 102-mm tall pipet tips (volume capacity of 1,250 µl) 8251 Protocol # PT3631-1 www.clontech.com Clontech Laboratories, Inc. Version # PR24221 19