96-well PCR Cleanup Manual for Non-Kit Users

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1 96-well PCR Cleanup Manual for Non-Kit Users

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3 Contents Introduction Microplate Components Required Buffer Formulations Equipment Required PCR Cleanup Procedure Overview of Procedure Sample Preparation Vacuum Filtration Protocol Centrifugation Protocol PCR Cleanup Automation Product Performance Troubleshooting Additional Accessories

4 Introduction The 96-well PCR Cleanup Kit provides a rapid method for purification of PCR products from unwanted reaction components. PCR products from 100bp to 10kb can be separated from nucleotides, salts, primers and proteins in less than 10 minutes without the need for time-consuming precipitations or labor intensive resin purifications. The purified DNA is ready to use in downstream applications such as fluorescent sequencing, restriction digestions, ligations, and microarray analysis. Microplate Components Required Catalog No. Description Qty/Case DNA Binding Plate µl V-bottom Collection Plate ml Waste Collection Plate well Adhesive Plate Seals 100 Buffer Formulations Buffer Qty needed Formulation (per plate) DNA Binding Buffer 20 ml 4M Guandine-HCI, 0.75 M Potassium Acetate, ph 4.6 Wash Buffer 10 ml 40% (v/v) 100 mm Tris, 20 mm EDTA, 0.4M NaCl, ph 7.5. Requires addition of ethanol prior to use. Wash Buffer to yield a final concentration of 60% (v/v) ethanol. Elution Buffer 10 ml 10 mm Tris, 0.1 mm EDTA, ph 7.5 All buffers listed in the table above are stable for 1 year at room temperature (15-30 C) Equipment Required Vacuum Filtration Protocol Appropriate pipettes and pipette tips Whatman Vacuum Manifold System (Cat. No contains manifold with gauge, regulator, two-way control valve and Teflon / silicone vacuum assist) or equivalent system compatible with Whatman plates 750 µl Large Spacer Plate (Cat. No or equivalent) Sufficient tubing and moisture trap Vacuum source capable of at least -15 inches Hg Pressure Conversion Chart Conversions from inches Hg to: Multiply by: Millibars (mbar) Millimeters of mercury (torr) 25.4 Kilopascals (kpa) Pounds per square inch (psi)

5 Equipment Required (Continued) Centrifugation Protocol Appropriate pipettes and pipette tips Centrifuge capable of 1800 x g Microplate carrier capable of accommodating 7.5 cm stack height Precautions This procedure involves the handling of amplification products. Proper care must be taken to avoid cross contamination of samples and buffers. Change gloves often. Change pipette tips after any contact with amplified material to avoid contamination of buffers or samples. Some of the buffers listed in this manual are potential eye/skin irritants. Proper laboratory precautions should be followed. This procedure is designed for research use only; not for use in diagnostic procedures. MSDS sheets are available upon request. Whatman plates are designed to be disposable, single-use-only products. 5

6 96-Well PCR Cleanup Procedure This section includes all of the information necessary to carry out the purification of PCR products using vacuum filtration and centrifuge systems. The protocol also can be adapted for use on liquid handling systems. Overview of Procedure PCR Product Add Binding Buffer Bind Filter Add Wash Buffer Wash Filter Add Elution Buffer Elute Filter and Collect 6

7 Sample Preparation Sample Volumes less than 25 µl Add enough DNA Binding Buffer to bring the total volume to 75 µl and mix by aspiration with a pipette. For example, to a 5 µl PCR reaction, add 70 µl DNA Binding Buffer. Sample Volumes between 25 µl and 125 µl (recommended) Add 2 volumes DNA Binding Buffer to 1 volume of the PCR sample and mix by aspiration with a pipette. Sample Volumes between 125 µl and 200 µl Add 1 volume of DNA Binding Buffer to 1 volume of the PCR sample and mix by aspiration with a pipette. Sample Volumes greater than 200 µl Divide the reaction into 2 or more tubes and follow the above criteria for sample preparation based on the new sample volume. Note: It is not necessary to remove mineral oil from the PCR samples. If mineral oil is being used, base the above calculations on the aqueous sample volume only. Vacuum Filtration Protocol 1. Prepare vacuum manifold for DNA binding. Attach the vacuum source to the manifold. Place the 2 ml Waste Collection Plate inside the vacuum manifold. Place the vacuum manifold collar on top of the manifold. Place the DNA Binding Plate on top of the vacuum collar, ensuring that the two plates are aligned alphanumerically (A1 to A1). 2. Apply the full volume of each prepared sample directly to the center of each well of a DNA Binding Plate. Apply vacuum between -15 and -20 inches Hg and allow the sample to be pulled through the filter media for 1 minute. Release the vacuum using the manifold switch and check to see that all liquid has been evacuated from each well. If fluid remains in any of the wells, repeat this step until all liquid has cleared. Note: If all 96-wells of the plate are not being processed simultaneously, the unused wells must be sealed with the vacuum assist or adhesive sealing tape before vacuum is applied. 7

Note: Ensure that ethanol has been added to Wash Buffer. If ethanol has not been added, refer to Buffer Formulation. 3. Add 400 µl Wash Buffer to each well of the DNA Binding Plate.

8 Note: Ensure that ethanol has been added to Wash Buffer. If ethanol has not been added, refer to Buffer Formulation. 3. Add 400 µl Wash Buffer to each well of the DNA Binding Plate. Apply vacuum between -15 and -20 inches Hg, and allow the buffer to be pulled through the filter media for 1 minute. Release the vacuum using the manifold switch, and check to see that all liquid has been evacuated from each well. If fluid remains in any of the wells, repeat this step until all liquid has cleared. 4. Apply maximum vacuum for an additional 2 minutes to dry the DNA Binding Plate. This step removes residual Wash Buffer from the DNA Binding Plate. Seal any unused wells of the DNA Binding plate before drying. Ensure that the wells in use are exposed to ambient airflow for efficient drying. Release the vacuum using the manifold switch. Using a clean absorbent paper towel, blot the bottom of the DNA Binding Plate to remove any residual Wash Buffer from the drip directors. The removal of ethanol present in the Wash Buffer is important for downstream applications. 5. Prepare vacuum manifold for DNA collection. As illustrated, place the 750 µl large Spacer Plate inside the vacuum manifold. Place a 250 µl Collection Plate on top of the spacer plate. Place the vacuum collar on top of the manifold. Place the processed DNA Binding Plate on top of the vacuum collar. Ensure that the DNA Binding Plate and the 250 µl Collection Plate are aligned alphanumerically (A1 to A1). 8

9 6. Add 100 µl Elution Buffer (or nuclease-free water) to the center of each well of the DNA Binding Plate. Allow the plate to stand for 1 minute. Apply vacuum between -15 and -20 inches Hg, and allow the buffer to be pulled through the filter media for a minimum of 1 minute. Release the vacuum using the manifold switch and ensure that all liquid has been evacuated from each well. If fluid remains in any of the wells repeat this step until all liquid has cleared. Note: If more concentrated eluate is desired, the volume of elution buffer can be decreased to a minimum of 25 µl. However, as elution volume is decreased, some loss in volume might be observed due to liquid retention in the filter media. 7. Remove the 250 µl Collection Plate from the vacuum manifold. The purified PCR product is now suitable for use in downstream applications. For storage, cover the 250 µl Collection Plate with the 96-well Adhesive Plate Seal. Centrifugation Protocol When using the centrifugation protocol outlined below, ensure that the centrifuge meets the criteria noted in the Equipment Required section. Refer to manufacturer s manual for operating instructions. 1. Prepare plates for DNA binding. Place the 96-well DNA Binding Plate on top of the 2 ml Waste Collection Plate to form a plate stack. Ensure that the two plates are aligned alphanumerically (A1 to A1). 2. Apply the full volume of each prepared sample directly to the center of each well of the DNA Binding Plate. Place the stacked plates in the centrifuge tray. 3. Centrifuge for 2.5 minutes at 1800 x g. The DNA is now bound to the filter media of the DNA binding plate. Remove the stacked plates from the centrifuge tray. 9

10 Note: Ensure that ethanol has been added to the Wash Buffer. If ethanol has not been added, refer to Buffer Formulation. 4. Add 400 µl Wash Buffer to each well of the DNA binding plate. Place the stacked plates in the centrifuge tray. 5. Centrifuge for 4.5 minutes at 1800 x g. This step removes the residual Wash Buffer from the DNA Binding Plate. Remove the stacked plates from the centrifuge tray. Using a clean absorbent paper towel, blot the bottom of the DNA Binding Plate to remove any residual Wash Buffer from the drip directors. The removal of ethanol present in the Wash Buffer is important for downstream applications. 6. Prepare plates for DNA Collection. Place the DNA Binding Plate on top of a 250 µl Collection Plate to form a plate stack, ensuring that the two plates are aligned alphanumerically (A1 to A1). 7. Add 100 µl Elution Buffer (or nuclease-free water) to the center of each well of the DNA Binding Plate. Allow the plates to stand for 1 minute. Place the stacked plates in the centrifuge tray. Note: If more concentrated eluate is desired, the volume of Elution Buffer can be decreased accordingly. However, as elution volume is decreased, some loss in volume might be observed due to liquid retention in the filter media. 8. Centrifuge for 2.5 minutes at 1800 x g. The PCR product has now been eluted into the 250 µl Collection Plate. 9. Remove the stacked plates from the centrifuge. The purified PCR product is now suitable for use in downstream applications. For storage, cover the Collection Plate with the 96-well Adhesive Plate Seal. PCR Cleanup Automation The Whatman 96-well PCR Cleanup System is compatible with high-throughput platforms. Automated protocols for specific high throughput systems can be obtained through Whatman technical service or by download at 10

11 Product Performance The 96-well PCR Cleanup Kit provides an easy-to-use method for purifying PCR products from unincorporated reaction components. After DNA is captured onto a filter media, impurities are washed away and the purified PCR product is eluted into an isotonic buffer. Eluted DNA is free of polymerases and other protein adjuncts (>99.5% removal), as well as nucleotides, salts and primers (see figure 1). The highly purified product is ready for use in applications such as fluorescent sequencing, restriction digestion, ligation, and microarray analysis without the need for further manipulations. High recovery of DNA (>85% - see figure 2) has been observed for PCR products ,000 bp in length with extremely reproducible results (less than 10% CV) and no cross contamination between wells. The kits are designed for vacuum filtration, centrifugation, and liquid handling systems, such as the Biomek 2000 instrument. Figure 1. Primer Removal 1.3Kb Globin Before After Before After Cleanup Cleanup Cleanup Cleanup MW 213bp Amelogenin Primer Bands Product from two PCR reactions (1.3kb -globin PCR, and 213bp Amelogenin PCR) visualized on a 2% agarose gel before and after purification using the 96-well PCR Cleanup Kit. MW = Promega PCR MW markers (1000, 750, 500, 300, 150 & 50 base pairs) Figure 2. PCR Product Size vs. Percent Recovery Average % Recovery 100% 90% 80% 70% 60% 50% PCR Product Size (numbers of base pairs) Different size PCR products processed using the 96-well PCR Cleanup Kit. Each fragment size was run n=8. Average percent recovery determined by PicoGreen assay. 11

12 Troubleshooting Problem Low DNA Yield Comments/Suggestions PCR reaction not optimized. PCR optimization studies can increase amplification efficiency for improved yield. Insufficient mixing with DNA Binding Buffer before transferring sample to the DNA Binding Plate. Sample not added to the center of the well. Sample that is not in contact with the filter media will not bind, resulting in low DNA recovery. Elution buffer not added to the center of the well. To ensure uniform coverage of the filter media, Elution Buffer should be added directly to the center of the filter media. Elution volume less than 50 µl. Due to liquid retention in the filter media, a higher percentage of volume loss will occur with lower elution volumes. Elution volumes less than 50 µl are only recommended if very high DNA concentrations are required. Nucleases present in the eluate. If using an alternative elution buffer (i.e. water), ensure that it is nuclease-free to ensure DNA integrity. 12

13 Problem Wells Not Filtering Comments/Suggestions Nonuniform airflow. If processing less than 96 wells simultaneously, the Vacuum Assist or sealing tape must be used on empty wells to ensure proper vacuum. Airlock due to bubble in the well. Buffers should be added to the center of each well to avoid air bubbles. Removal of air bubbles by sample agitation is recommended. Insufficient vacuum. Verify that the vacuum manifold reaches the specified vacuum as outlined in the protocol. Poor DNA Performance Ethanol in Eluate High Salt in Eluate Unexpected Size Fragments on Gel Incomplete drying. Ensure that the Wash Buffer drying process is followed carefully and completely. Ensure that the wash step has been properly performed. Verify that ethanol has been added to Wash Buffer as outlined in Preparation of Buffer. PCR reaction not optimized. PCR optimization can increase priming specificity for the desired product. Sample cross contamination or buffer contamination. Proper molecular biology precautions must be maintained when handling amplified products. All pipette tips must be changed after contacting amplification products. 13

14 Additional Accessories Catalog No. Description Qty/Case Vacuum Manifold System (contains: Manifold with gauge, regulator, two-way control valve, and Teflon /silicone vacuum assist) 1 per/box Teflon/Silicone Vacuum Assist (replacement for vacuum assist contained in Vacuum Manifold System) 6 per/pack ml Waste Collection Plate 25 per/case well Filtrate Directors (for Biomek 2000 processing) 25 per/case Small Whatman Vacuum Adapter Collar for Biomek per/pack Medium Whatman Vacuum Adapter Collar for Biomek per/pack Notice The information in this document is given in good faith, and is for guidance only. It is not intended to override or void any of Whatman s Standard Conditions of Sale. Those Conditions remain in full and complete effect, and govern all relations between Whatman and the purchaser of the goods which relate to this document. PCR is a patented process covered by U. S. Patents 4,683,195 and 4,683,202 and foreign equivalents owned by Hoffman-La Roche AG. PicoGreen is a registered trademark of Molecular Probes, Inc. Biomek is a registered trademark of Beckman Coulter, Inc. Teflon is a registered trademark of DuPont Whatman is a registered trademark of Whatman Inc. Copyright, Whatman Inc., 2002 Printed in USA 14

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16 Whatman For pricing and availability, please contact: USA Whatman Inc. 9 Bridewell Place Clifton, NJ Toll Free: 800-WHATMAN Fax: info@whatman.com Europe/Middle East/Africa Whatman International Ltd. Whatman House St. Leonard s Road 20/20 Maidstone Kent, England ME 16 OLS Tel: +44 (0) Fax: +44 (0) info@whatman.com China Whatman Shanghai Liaison Office No. 8 Dong An Room 909 Pine City Hotel Shanghai P.R.China Tel: Fax: wmansha@online.sh.cn Japan Whatman Japan Ltd. Daiwa Ueno Building 1F 6-10 Ueno 5-chrome, Taito-ku Tokyo Japan Tel: Fax: japaninfo@whatman.co.jp Asia Pacific Whatman Asia Pacific Pte Ltd. 171 Chin Swee Road #08-01 San Centre Singapore Tel: Fax: wap@whatman.com.sg India Whatman India Liaison Office 503 Swastik Chambers U.B.Chowk V.N.Purav Marg, Chembur Mumbai India Tel: Fax: whatmanb@bom3.vsnl.net.in The Advantages Filter Through SM 9 Bridewell Place, Clifton, NJ Part No. 69I , Rev. A

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