GENECLEAN Turbo 96 System Qbiogene s Optimized Kit for DNA Purification in a 96 well Format

Transcription

1 Qbiogene s Optimized Kit for DNA Purification in a 96 well Format Revision # E30

2 GENECLEAN Turbo 96 System Qbiogene s Optimized Kit for DNA Purification in a 96 well Format Application Manual Revision # E30 Product Description # of Preps Cat No. GENECLEAN Turbo 96 1 x 96 preps x 96 preps x 96 preps Any Questions? Call Technical Support at (800)

3 TABLE OF CONTENTS 1. GENECLEAN Turbo 96 Introduction 1.1 Related BIO 101 brand products 2. GENECLEAN Turbo 96 System 2.1 Background to the GENECLEAN Turbo 96 System 2.2 Key Features & Applications 3. GENECLEAN Turbo 96 Product Line 3.1 GENECLEAN Turbo 96 Components Catalog # (1 x 96 preps) Catalog # (4 x 96 preps) Catalog # (10 x 96 preps) 3.2 Shipping and Storage of the GENECLEAN Turbo 96 Kit 4. GENECLEAN Turbo 96 Protocol 4.1 Equipment & Reagents required for the GENECLEAN Turbo 96 Protocol Equipment Reagents not provided with the GENECLEAN Turbo 96 Kit 4.2 Things to know before using the GENECLEAN Turbo 96 Protocol Preparation of the Turbo 96 Wash Solution Binding Capacity Volume 4.3 GENECLEAN Turbo 96 Kit Detailed Protocol 4.4 GENECLEAN Turbo 96 Kit Short Protocol 5. Troubleshooting 5.1 Low Plasmid Recovery 5.2 Trouble with Downstream Applications 6. Appendices i) Examples of PCR and genomic DNA purification ii) Product Use Limitation & Warranty 4 visit us on the web at

4 1. GENECLEAN Turbo 96 Introduction The GENECLEAN Turbo 96 is a high-throughput DNA purification kit optimized for speed, easy of use, and the best DNA quality. This is made possible by the use of a proprietary GENECLEAN GLASSMILK embedded membrane filter plate. This filter plate efficiently binds DNA in the range of 100bp to 300kb in size. GENECLEAN Turbo 96 is used to clean PCR products and purify DNA from enzymatic and isolation processes. The kit contains a detailed protocol and all the reagents needed to successfully purify DNA in 96 well format. The purified DNA is ideal for PCR, sequencing, DNA arrays, and other high-throughput applications. 1.1 BIO 101 Related Products Plasmid DNA Isolation in 96well format Product Description # of Preps Cat. # RPM Turbo 96* 1 x 96 preps x 96 preps x 96 preps well Prep Express** 4 x 96 preps x 96 preps * Contains all solutions/materials needed to successfully isolate plasmid DNA from bacteria in 96 well format. ** Contains DNA Filter Plate and Binding Solution. This economical kit provides all formulas necessary to make your own solutions. Purification of PCR Products in 1 step for fluorescent cycle sequencing Product Description # of Preps Cat. # SeqDirect PCR Cleaning Kit 16 preps preps preps preps preps Purification of PCR Products in 96 well format for fluorescent cycle sequencing Product Description # of Preps Cat. # SeqDirect 96 PCR Cleaning Kit 1 x 96 preps Any Questions? Call Technical Support at (800)

5 2. GENECLEAN Turbo 96 System 2.1 Background to the GENECLEAN Turbo 96 System The GENECLEAN Turbo 96 Kit is a high-throughput system that uses the patented Turbo system. The Turbo system consists of the GENECLEAN GLASSMILK embedded membrane 96 well filter plates, which has been optimized for DNA purification from any type of agarose gel or solution in the size range of 100bp to 300kb. 2.2 Key Features & Applications Novel GLASSMILK embedded 96 well filter plates 1 plate processed in ~45 minutes Purification of DNA Fragments in the range of 100bp to 300kb Clean PCR Products Purify DNA from enzymatic and isolation processes DNA eluted in 50µl of H 2 0 and is ready-to-use in any downstream application Up to 10µg of DNA per well No alcohol precipitation 3. GENECLEAN Turbo 96 Product Line The GENECLEAN Turbo 96 Kit comes as a complete package containing all components and protocols needed for 1 x 96 preps, 4 x 96 preps, and 10 x 96 preps. Product Description # Preps Cat. # GENECLEAN Turbo 96 Kit 1 x 96 preps x 96 preps x 96 preps GENECLEAN Turbo 96 Components Catalog # (1 x 96 preps) Product Volume GENECLEAN Turbo 96 DNA Filter Plate 1 GENECLEAN Turbo 96 Binding Solution* 75 ml Turbo 96 Wash Solution* 2 18 ml Collection Plate 1 Collection Plate Mat 1 Elution Solution* 3 10 ml * Contains guanidine thiocyanate, use with proper precaution *2 The GENECLEAN Turbo 96 Wash Solution is prepared by adding the provided solution to predetermined amounts of 100% ethanol as per Table 1 on page 8. *3 RNase/DNase/pyrogen-free water 6 visit us on the web at

6 3.1.2 Catalog # (4 x 96 preps) Product Size/Volume GENECLEAN Turbo 96 DNA Filter Plate 4 GENECLEAN Turbo 96 Binding Solution* 300 ml Turbo 96 Wash Solution* 2 72 ml Collection Plate 4 Collection Plate Mat 4 Elution Solution* 3 40 ml * Contains guanidine thiocyanate, use with proper precaution *2 The GENECLEAN Turbo 96 Wash Solution is prepared by adding the provided solution to predetermined amounts of 100% ethanol as per Table 1 on page 8. *3 RNase/DNase/pyrogen-free water Catalog # (10 x 96 preps) Product Size/Volume GENECLEAN Turbo 96 DNA Filter Plate 10 GENECLEAN Turbo 96 Binding Solution* 750 ml Turbo 96 Wash Solution* 2 2 x 90 ml Collection Plate 10 Collection Plate Mat 10 Elution Solution* ml * Contains guanidine thiocyanate, use with proper precaution *2 The GENECLEAN Turbo 96 Wash Solution is prepared by adding the provided solution to predetermined amounts of 100% ethanol as per Table 1 on page 8. *3 RNase/DNase/pyrogen-free water 3.2 Shipping and Storage of the GENECLEAN Turbo 96 Kit The GENECLEAN Turbo 96 Kit is shipped and stored at room temperature. It is stable for at least 1 year from the time it is shipped. Any Questions? Call Technical Support at (800)

7 4. GENECLEAN Turbo 96 Protocol 4.1 Equipment and reagents required for the GENECLEAN Turbo 96 Protocol Equipment Multi-channel pipette Vacuum manifold Vacuum aspirator Centrifuge/rotor for deep 96 well plates Standard and 1-2 ml 96 well plates Reagents Not Provided with the GENECLEAN Turbo 96 Kit Ethanol 4.2 Things to Know Before Using the GENECLEAN Turbo 96 Protocol Preparation of the Turbo 96 Wash Solution Before using, add the Turbo 96 Wash Solution to the recommended amount of ethanol as per Table 1 below. Close the cap tightly after use. 100% Alcohol Cat # # of Preps Wash Solution (200 proof) x ml 162 ml x ml 648 ml x 96 2 x 90 ml 810 ml per 90 ml Binding Capacity Up to 10 µg of DNA per well Volume Up to 800 µl per well. When dispensing solutions keep the pipette tips as close as possible to the wells but outside the well space to avoid sample overflow due to solution displacement by the tips. GENECLEAN Turbo 96 DNA Filter Plate fits all 96 well vacuum manifolds. The specially designed long drip director system prevents sample cross-contamination during vacuum filtration and centrifugation. The GENECLEAN Turbo 96 Kit can be used with any vacuum source, i.e. vacuum pump, house vacuum or water aspirator. 8 visit us on the web at

8 4.3 GENECLEAN Turbo 96 Kit Protocol! = Read Me First 1. Add 600µl of GENECLEAN Turbo 96 Binding Solution to DNA samples.! DNA Samples Up to 100µl PCR Reactions Up to 10µg DNA from enzymatic/isolation processes in = 200µl. It might be necessary to transfer samples to a deep 96 well plate before adding the Binding Solution. 2. Mix the DNA/Binding Solution by pipetting up and down a couple of times and transfer to GENECLEAN Turbo 96 DNA Filter Plate assembled into a vacuum manifold with a waste tray in the bottom. Incubate 5 minutes at room temperature and drain the supernate by vacuum filtration for 2-3 minutes or until all the wells are dry.! It is normal for the DNA/Binding solution to start dripping during the 5-minute incubation step.! The vacuum filtration process will proceed faster if unused wells are sealed (i.e. with parafilm cover paper.) Do not use the parafilm sheet as it can pop during vacuum filtration depending on the vacuum source. Sealing is automatically achieved by placing the paper on top of unused wells and applying vacuum. Do not cover wells containing your samples.! If a well clogs, gently stir contents with a clean 200 µl pipette tip while gently touching the membrane surface during the filtration process. A stronger vacuum source may be necessary if clogging persists. 3. Add 700 µl of Turbo 96 Wash Solution per used well (Add ethanol before first use, see page 8) and apply vacuum for ~1 minute or until all the wells are dry. Repeat the wash step one more time. After the second wash has drained keep the vacuum on for an additional 10 minutes. 4. Remove the GENECLEAN Turbo 96 DNA Filter Plate from the vacuum manifold and align on top of a standard 96 well plate. Spin at 2000 x g for 10 minutes at room temperature (~3000 rpm in IEC Centra CP8R centrifuge) to remove all traces of ethanol from the membrane and drip director tips.! It is not necessary to secure the DNA Filter Plate and standard 96 well plate as long as the combined height is less than or equal to the side wall height of the 96 well rotor. 5.. Elute the DNA with Elution Solution, H 2 O or TE by centrifugation while keeping track of orientation to avoid sample mix-up. Align the GENECLEAN Turbo 96 DNA Filter Plate on top of a Collection Plate. Add 50 µl of Elution Solution DIRECTLY on to the GLASSMILK embedded membrane, incubate 5 minutes at room temperature and spin 2 minutes at 2000 x g to collect the DNA. DNA is ready for use. Store at 4 C or -20 C Any Questions? Call Technical Support at (800)

9 6. The Collection Plate can be sealed with the provided Collection Plate Mat. This is best achieved by aligning the cover over the Collection Plate and applying pressure with the side of a microtube rack to seal the wells. Alternatively, a high-throughput sealing device is available through Fisher Scientific (Cat. # )! Spin the Collection Plate prior to removing the Collection Plate Mat. Condensation may form on the inside cover causing a change in sample concentration. Cross-contamination between samples could occur if the sealed plate has been inverted.! To remove the Collection Plate Mat hold the Collection Plate securely on the bench top and pull the mat by the tab in a diagonal fashion (i.e. toward the opposite side of the tab.) 4.4 GENECLEAN Turbo 96 Short Protocol A. DNA Binding/Washing 1. Add 600 µl GENECLEAN Turbo 96 Binding Solution to DNA samples. 2. Mix and transfer to GENECLEAN Turbo 96 Filter Plate assembled into a vacuum manifold with a waste tray in the bottom. Incubate 5 minutes and apply vacuum until all the wells are dry (2-3 minutes.) 3. Add 700 µl of Turbo 96 Wash Solution and filter until dry (< 1 minute.) Repeat the wash. B. Removal of Trace Wash Solution from DNA Filter 4. Keep the vacuum source on for 10 minutes after the second wash. 5. Align the GENECLEAN Turbo 96 DNA Filter Plate on top of a standard 96 well plate and spin for 10 minutes at 2000 x g. C. DNA Elution 6. Align the GENECLEAN Turbo 96 DNA Filter Plate on top of Collection Plate. Add 50 µl H 2 O or TE DIRECTLY on to the GLASSMILK embedded membrane, incubate for 5 minutes and spin for 2 minutes at 2000 x g to collect the DNA. Cover Collection Plate with Collection Plate Mat. DNA is ready for use. 5. Troubleshooting 5.1 Low DNA recovery Make sure that the DNA and GENECLEAN Turbo 96 Binding Solution are mixed properly incubated before filtration (step 2.) Make sure that ethanol has been added to the Turbo 96 Wash Solution before first use (step 3.) Keep the wash bottle tightly capped after each use. Add Elution Solution, H 2 O or TE DIRECTLY on to the GLASSMILK embedded membrane and let it soak for about 5 minutes before collecting the DNA (step 5.) 10 visit us on the web at

10 5.2 Trouble with Downstream Applications Inefficient removal of ethanol from the DNA Filter Plate before Elution Make sure that the vacuum source is kept on for 10 minutes after the second wash (step 3.) Make sure that the washed plate is spun for 10 minutes at step 4 before eluting the DNA. Aerate the sample for ~5-10 minutes before using to allow any residual ethanol to evaporate. 6. Appendices (i) Examples of PCR and genomic DNA Purification Lane 1: 1 kb ladder Lane 2: 1.5 kb PCR fragment Lane 3: 500 bp PCR fragment Lane 4: 780 bp PCR Fragment a: before purification b: after purification c: purified product cut with EcoRI d: purified product cut with PvuI Lane 1: 1 kb Ladder Lane 2: 100 bp PCR fragment Lane 3: 150 bp PCR fragment Lane 4: 200 bp PCR Fragment a: before purification b: after purification Lane 1: Lambda HindIII markers Lane 2: Rat liver genomic DNA a: before purification b: after purification c: purified product cut with BamHI Any Questions? Call Technical Support at (800)

11 (ii) Product Use Limitation & Warranty Unless otherwise indicated, this product is for research use only. Purchase of Qbiogene products does not grant rights to reproduce, modify, repackage the products or any derivative thereof to third parties. Qbiogene makes no warranty of any kind, expressed or implied including merchantability or fitness for any particular purpose, except that the products sold will meet our specifications at the time of delivery. Buyer s exclusive remedy and Qbiogene s sole liability hereunder shall be limited to, at our option, product credits, refund of the purchase price of, or the replacement of all material(s) that does not meet our specification. By acceptance of the product, Buyer indemnifies and holds Qbiogene harmless against, and assumes all liability for the consequence of its use or misuse by the Buyer, its employees or others, including, but not limited to, the cost of handling. Said refunds or replacement is conditioned on Buyer notifying Qbiogene within thirty (30) days of receipt of product. Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by the Buyer of all claims hereunder with respect to said material(s). GENECLEAN products are covered under US patent #6,027,750. GENECLEAN, GLASSMILK, and BIO 101 are registered trademarks of Qbiogene. 12 visit us on the web at

12 Notes Any Questions? Call Technical Support at (800)

13 Notes 14 visit us on the web at