GFX PCR DNA and Gel Band Purification Kit

Transcription

1 instructions product code: GFX PCR DNA and Gel Band Purification Kit Warning For research use only. Not recommended or intended for the diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. Harmful i Ed. AA

2 Handling Storage Store at ambient temperature. Page finder Components of the kit Quality control Materials not supplied World Wide Web address Safety warnings and precautions Introduction Protocols Essential preliminaries Purification of DNA from solution Purification of DNA from gel bands Appendixes 1. Protocol for MicroPlex 24 Vacuum Protocol for MicroPlex 24 (Spin) Troubleshooting References Companion products Licenses Trademarks p2

3 Quality control GFX PCR DNA and Gel Band Purification Kit is tested for the ability to quantitatively isolate a 910 base-pair PCR product from solution and from an agarose gel band. Materials not supplied Reagents Absolute Ethanol Elution Buffer 10 mm Tris-HCl, ph 8.0 or TE buffer (10 mm Tris-HCl, ph 8.0, 1 mm EDTA) or autoclaved double-distilled water. Equipment Microcentrifuge that accomodates 1.5 ml microcentrifuge tubes. Tubes 1.5 ml microcentrifuge tubes Scalpel or razor blade Incubator or water bath 60 C Components of the kit The following components are included in this product: Capture buffer GFX columns Collection tubes Wash buffer* Buffered solution containing acetate and chaotrope. MicroSpin columns pre-packed with a glass fiber matrix. 2 ml capless microcentrifuge tubes. Tris-EDTA buffer (10 mm Tris-HCl ph 8.0, 1mM EDTA). Add absolute ethanol to a final concentration of 80% before use as indicated in the note below. *The wash buffer requires dilution before use. To the bottle containing wash buffer, add 48 ml of absolute ethanol. Mix by inversion. After diluting this component, indicate on the label that this step has been completed. When not in use, store the wash buffer-ethanol mixture AIRTIGHT at ambient temperatures. p3

4 World Wide Web address Visit the GE Healthcare home page for regularly updated product information. Safety warnings and precautions Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. All chemicals should be considered as potentially hazardous. Only persons trained in laboratory techniques and familiar with the principles of good laboratory practice should handle these products. Suitable protective clothing such as laboratory overalls, safety glasses, and gloves should be worn. Care should be taken to avoid contact with skin or eyes; if contact should occur, wash immediately with water (see Material Safety Data Sheet for specific recommendations). Warning: This protocol requires the use of ethanol and formamide. Gel reagents may contain acrylamide, a neurotoxin and suspected carcinogen. Please follow the manufacturer s Material Safety Data Sheet regarding safe handling and use of these materials. p4

5 Introduction GFX PCR DNA and Gel Band Purification Kit uses a chaotropic agent that denatures protein, dissolves agarose, and promotes the binding of double-stranded DNA (100 base-pairs to 48 kilobase-pairs) to a glass fiber matrix (2, 3). Once the DNA is captured, proteins and salt contaminants are washed away, and the purified DNA is eluted in a low ionic strength buffer (TE, Tris-HCl or water). DNA samples are recovered in a concentrated form by eluting with as little as 10 µl of buffer or water. GFX PCR DNA and Gel Band Purification Kit can be used to purify DNA (e.g., PCR products, restriction fragments) from solution and from both TAE and TBE agarose gel bands. Typical recoveries are >80% from solution and > 60% from gel bands. GFX purification of PCR products from solution removes 99.5% of primers and free nucleotides and can be performed in less than 5 minutes. DNA purification from a gel band can be completed in just 15 minutes. Purified DNA can be used directly without an alcohol precipitation in a variety of applications including PCR amplification, restriction digest analysis and subcloning. To efficiently process 24, 48 or 96 samples simultaneously, the kit can be used with MicroPlex 24 Vacuum or MicroPlex 24 (see Appendixes 1 and 2, respectively). Use with MicroPlex 24 Vacuum can result in time savings of >20% for 48 samples and >40% for 96 samples as compared with microfuge based purification. p5

6 The basic protocol for the GFX PCR DNA and Gel Band Purification Kit involves the following steps: Step Comments Component Denaturation/ Proteins are denatured. For gel band Capture buffer solubilization purification, agarose is dissolved. Capture The sample is passed through the GFX column GFX column to capture the DNA onto the glass fiber matrix. Washing/ Matrix-bound DNA is washed with Wash buffer Drying an ethanolic buffer to remove salts and other contaminants. This step also dries the matrix prior to elution. Elution The purified DNA is eluted from the Not supplied GFX column in low ionic strength buffer (10 mm Tris-HCl ph 8.0, TE ph 8.0 or water). p6

7 Protocols Essential Preliminaries To elute the purified plasmid DNA, we recommend using either 10 mm Tris-HCl ph 8.0, TE buffer (10 mm Tris-HCl ph 8.0, 1 mm EDTA) or autoclaved double-distilled water. Prior to the first use, the wash buffer must be diluted (see page 3). After diluting, indicate on the label that this step has been completed. Purification from solution or from gel bands Procedures 1 and 2 provide protocols for purifying PCR products, restriction fragments and plasmid DNA from solution or from agarose gel bands, respectively. Double-stranded DNA can be purified from solution volumes of up to 100 µl and from gel slices of up to 300 mg. Note that no modifications are required for purification from gels run in boratebased buffers (e.g., TBE). Eluting the DNA in a concentrated form To recover DNA in a concentrated form, use elution volumes that are less than the volume of the sample being purified. For optimal recovery use volumes 50 µl. Using elution volumes less than 50 µl will reduce the yield. For example DNA eluted in 30 µl will concentrate the recovered DNA approximately 1.5 Fold with a loss of 20 30% as compared to a 50 µl elution volume. An elution with 10 µl will concentrate the DNA recovered approximately 2.5 Fold with a loss of 60 70%. p7

8 Purification of DNA from solution ➊ The maximum volume of PCR reaction/dna solution that can be processed with the following protocol is 100 µl. 1.1 Place one GFX column in a collection tube for each purification to be performed. 1.2 Add 500 µl of capture buffer to the GFX column. 1.3 Transfer the DNA solution (up to 100 µl) to the GFX column. If purifying a PCR sample, avoid transferring mineral oil to the column. 1.4 Mix thoroughly by pipetting the sample up and down 4 6 times. 1.5 Centrifuge in a microcentrifuge at full speed for 30 s. 1.6 Discard the flow-through by emptying the collection tube. Place the GFX column back inside the collection tube. 1.7 Add 500 µl of wash buffer to the column. Centrifuge at full speed for 30 s. 1.8 Discard the collection tube and transfer the GFX column to a fresh 1.5 ml microcentrifuge tube (i.e. NOT a collection tube). 1.9 Apply 50 µl of elution buffer (10 mm Tris-HCl ph 8.0, TE ph 8.0 or autoclaved double-distilled water) directly to the top of the glass fiber matrix in the GFX Column. To elute DNA in a more concentrated form reduce the elution volume to no less than 10 µl. Note: Using low elution volumes to concentrate the DNA sample will reduce recovery (see page 7). 8

9 ➋ 1.10 Incubate the sample at room temperature for 1 min Centrifuge at full speed for 1 min to recover the purified DNA. Note: For 50 µl elutions, the actual volume of sample recovered will range from 40 to 50 µl. For a 10 µl elution, the recovered volume will range from 5 to 7 µl. Purification of DNA from gel bands The maximum weight of gel slice that can be processed with the following protocol is 300 mg; the column can accommodate a maximum volume of 600 µl (i.e., 300 mg of gel slice and 300 µl of capture buffer). 2.1 Weigh an empty 1.5 ml microcentrifuge tube to the nearest 10 mg and record the weight. 2.2 Using a clean razor blade or scalpel, excise the slice of agarose containing the DNA band to be purified. Cut as close to the DNA band as possible. Cut the slice into several smaller pieces and transfer them to the pre-weighed 1.5 ml microcentrifuge tube. 2.3 Weigh the tube containing the agarose slice to the nearest 10 mg, and subtract the weight of the empty tube to determine the weight of the slice. 2.4 To the gel slice add 10 µl of capture buffer for each 10 mg of gel slice (maximum column capacity is 300 µl of capture buffer added to a 300 mg gel slice). 2.5 Close the tube and mix by vortexing vigorously. Incubate at 60 C until the agarose is completely dissolved (5 15 min). p9

10 2.6 During the incubation, place one GFX column in a collection tube for each purification to be performed. 2.7 After the agarose is completely dissolved, centrifuge briefly to collect the sample at the bottom of the tube. 2.8 Transfer the sample to the GFX column. Incubate at room temperature for 1 min. 2.9 Centrifuge in a microcentrifuge at full speed for 30 s Discard the flow-through by emptying the collection tube. Place the GFX column back inside the collection tube Add 500 µl of wash buffer to the column. Centrifuge at full speed for 30 s Discard the collection t and transfer the GFX column to a fresh 1.5 ml microcentrifuge tube (i.e. NOT a collection tube) Apply 50 µl of elution buffer (10 mm Tris-HCl ph 8.0, TE ph 8.0 or autoclaved double-distilled water) directly to the top of the glass fiber matrix in the GFX column. To elute DNA in a more concentrated form reduce the elution volume to no less than 10 µl. Note: Using low elution volumes to concentrate the DNA sample will reduce recovery (see page 7) Incubate the sample at room temperature for 1 min Centrifuge at full speed for 1 min to recover the purified DNA. Note: For 50 µl elutions, the actual volume of sample recovered will range from 40 to 50 µl. For a 10 µl elution, the recovered volume will range from 5 to 7 µl. p10

11 Appendix 1: Protocol for MicroPlex 24 Vacuum Either MicroPlex 24 Vacuum or MicroPlex 24 may be used to efficiently process large numbers of samples simultaneously. If a vacuum source capable of generating ~200 mm Hg (e.g. a house vacuum) is available, we recommend that MicroPlex 24 Vacuum be used for high throughput purification. If a tabletop centrifuge and the appropriate microtiter plate rotor is available, MicroPlex 24 may be used. Using the MicroPlex 24 systems will result in significant time savings. For example, using MicroPlex 24 Vacuum can result in time savings of >20% for 48 samples and >40% for 96 samples as compared with microfuge-based purification. For either MicroPlex procedure, DNA recovery will be approximately 30% to 40% lower than that achieved with standard kit protocols. To use the MicroPlex 24 Vacuum, follow the MicroPlex 24 Vacuum Assembly steps below and then go to either Option A: Purification of DNA from solution (page 12) or Option B: Purification of DNA from gel bands (page 12) depending on the sample type. MicroPlex 24 Vacuum assembly 1. Use the Vacuum Applications instructions provided with the MicroPlex 24 to assemble the manifold and connect it to the vacuum source. 2. Place the required number of GFX columns in MicroPlex 24 manifold. p11

12 3. Fill any unused holes in the manifold with plugs included with MicroPlex 24 Vacuum. Note: Vacuum should NOT be applied to the manifold until ALL of the samples have been transferred to the columns and incubated. 4. Make sure that the vacuum line stopcock is in the closed position (i.e. perpendicular to the vacuum tubing) and that the manifold is placed squarely on the gasket and collection tray. This is necessary to ensure proper application of the vacuum. 5. Proceed to Option A if purifying DNA from solution, or alternatively use Option B if purifying from gel bands (see below). Option A: Purification of DNA from solution 1. Add 500 µl of capture buffer to each GFX column. 2. Transfer the DNA solution (up to 100 µl) to the GFX columns in the MicroPlex 24. If purifying a PCR sample, avoid transferring mineral oil to the column. 3. Proceed to DNA binding and recovery page 13. Option B: Purification of DNA from gel bands 1. Weigh an empty 1.5 ml microcentrifuge tube to the nearest 10 mg and record the weight. 2. Using a clean razor blade or scalpel, excise the slice of agarose containing the DNA band to be purified. Cut as close to the DNA band as possible. Cut the slice into several smaller pieces and transfer them to the pre-weighed 1.5 ml microcentrifuge tube. 3. Weigh the tube containing the agarose slice to the nearest 10 mg, and subtract the weight of the empty tube to determine the weight of the slice. p12

13 4. To the gel slice add 10 µl of capture buffer for each 10 mg of gel slice (maximum column capacity is 300 µl of capture buffer added to a 300 mg gel slice). 5. Close the tube and mix by vortexing vigorously. Incubate at 60 C until the agarose is completely dissolved (5 15 minutes). 6. After the agarose is completely dissolved, centrifuge briefly to collect the sample at the bottom of the tube. 7. Transfer the sample (up to 600 µl) to the GFX columns in the MicroPlex 24. Incubate at room temperature for 1 min. 8. Proceed to DNA binding and recovery, immediately below. DNA binding and recovery 1. Turn the vacuum supply (e.g. house vacuum) on at the source. Turn the stopcock in the vacuum tubing assembly to the open position (i.e. parallel to the tubing). After the samples have been drawn through all of the columns and into the collection tray, turn the stopcock to the closed position, leaving the vacuum supply on at the source. 2. Add 500 µl of wash buffer to each column. Turn the stopcock to the open position. Allow the solution to be drawn through each column and continue to apply the vacuum for 10 min to remove residual buffer and dry the matrix. Note: If using 2, 3 or 4 manifolds connected to the same vacuum source in parallel, dry min. Avoid over-drying (>30 min); DNA may become irreversibly bound to the glass fiber matrix. p13

14 3. Turn the stopcock to the closed position. Allow the vacuum pressure has fully dissipated, remove the manifold from the collection tray and place it on a clean paper towel. (The manifold should separate easily from the collection tray.) Discard the used collection tray and save the gasket. 4. For short-term storage, purified plasmid DNA can be collected using MicroPlex 24 Vacuum. (If desired, the trays containing the final samples can be covered with sealing tape from Costar, catalog no. 3095). Transfer the gasket and manifold to a new collection tray. Reassemble the MicroPlex 24 Vacuum system with the new collection tray. Proceed to the next step. Alternatively, the columns can be transferred to 1.5 ml microcentrifuge tubes for collection of samples using a microcentrifuge. This eliminates the need to transfer samples from the collection tray to tubes for long term storage. 5. Apply 125 µl of elution buffer (10 mm Tris-HCl ph 8.0 or TE ph 8.0 or autoclaved double-distilled water) directly to the top of the glass fiber matrix in the GFX column. 6. Incubate the sample at room temperature for 1 min. 7. To collect the purified DNA using MicroPlex 24 Vacuum, turn the stopcock to the open position. After all the samples have been drawn through the columns and into the collection tray, turn the stopcock to the closed position. Allow the vacuum pressure to dissipate for seconds and separate the collection tray (which contains the purified plasmid DNA) from the manifold as previously described. If desired, the tray containing the final samples can be covered with sealing tape from Costar (catalog no. 3095). p14

15 8. Alternatively, if using 1.5 ml microcentrifuge tubes, recover the DNA by centrifugation at full speed for 1 min in a microcentrifuge. Note: The recovered elution volume will be 40 60% of the applied volume for the MicroPlex 24 Vacuum method of sample recovery. DNA recovery will be approximately 30% to 40% lower than that from the microfuge procedure. Appendix 2: Protocol for MicroPlex 24 (Spin) Either MicroPlex 24 Vacuum or MicroPlex 24 may be used to efficiently process large numbers of samples simultaneously. If a vacuum source capable of generating ~200 mm Hg (e.g. a house vacuum) is available, we recommend that MicroPlex 24 Vacuum be used for high throughput purification. If a tabletop centrifuge and the appropriate microtiter plate rotor is available, MicroPlex 24 may be used. Using the MicroPlex 24 systems will result in significant time savings. For example, using the MicroPlex 24 Vacuum can result in time savings of >20% for 48 samples and >40% for 96 samples as compared to microfuge based methods. For either MicroPlex procedure, DNA recovery will be approximately 30% to 40% lower than with the microfuge procedures. To use the MicroPlex 24, follow the MicroPlex 24 assembly steps and then go to either Option A: Purification of DNA from solution (page 16) or Option B: Purification of DNA from gel bands (page 16) depending on the sample type. p15

16 MicroPlex 24 Assembly 1. Use the Spin Applications instructions provided with the MicroPlex 24 to assemble the manifold and V-bottom collection tray. 2. Place the required number of GFX columns in the MicroPlex 24 manifold. 3. Proceed to page 16 and use Option A if purifying DNA from solution, or alternatively use Option B if purifying from gel bands. Option A: Purification of DNA from solution 1. Add 500 µl of capture buffer to each GFX column. 2. Transfer the DNA solution (up to 100 µl) to the GFX columns in the MicroPlex 24 manifold. If purifying a PCR sample, avoid transferring mineral oil to the column. 3. Proceed to DNA binding and recovery page 17. Option B: Purification of DNA from gel bands 1. Weigh an empty 1.5 ml microcentrifuge tube to the nearest 10 mg and record the weight. 2. Using a clean razor blade or scalpel, excise the slice of agarose containing the DNA band to be purified. Cut as close to the DNA band as possible. Cut the slice into several smaller pieces and transfer them to the pre-weighed 1.5 ml microcentrifuge tube. 3. Weigh the tube containing the agarose slice to the nearest 10 mg, and subtract the weight of the empty tube to determine the weight of the slice. p16

17 4. To the gel slice add 10 µl of capture buffer for each 10 mg of gel slice (maximum column capacity is 300 µl of capture buffer added to a 300 mg gel slice). 5. Close the tube and mix by vortexing vigorously. Incubate at 60 C until the agarose is completely dissolved (5-15 min). 6. After the agarose is completely dissolved, centrifuge briefly to collect the sample at the bottom of the tube. 7. Transfer the sample (up to 600 µl) to the GFX columns in the MicroPlex 24. Incubate at room temperature for 1 min. 8. Proceed to DNA binding and recovery page 17. DNA binding and recovery 1. Centrifuge the MicroPlex 24 unit for 2 min at g(a minimum force of 700 g is needed). Note: The appropriate centrifugation speed for a specific rotor can be calculated from the following formula. RCF = (1.12)(r)(rpm/1 000) 2 where RCF = relative centrifugal force; r = radius in mm measured from the center of the spindle to the bottom of the rotor bucket; and rpm = revolutions per minute. 2. Add 500 µl of wash buffer to each column. Centrifuge the MicroPlex 24 unit for 10 min. p17

18 3. For short-term storage, purified DNA can be collected using MicroPlex 24 and a collection tray. If desired, the trays containing the final samples can be covered with sealing tape from Costar (catalog no. 3095). To collect samples using MicroPlex 24, remove the manifold from the collection tray and place it on a clean paper towel. Reassemble the MicroPlex 24 unit with a fresh collection tray and proceed to the next step. Discard the used collection tray. Alternatively, the columns can be transferred to 1.5 ml microcentrifuge tubes for collection of samples using a microcentrifuge. This eliminates the need to transfer samples from the collection tray to tubes for long-term storage. 4. Apply 100 µl of elution buffer (10 mm Tris-HCl ph 8.0 or TE ph 8.0 or autoclaved double-distilled water) directly to the top of the glass fiber matrix in the GFX column. 5. Incubate the samples at room temperature for 1 min. 6. To collect the purified DNA, centrifuge the MicroPlex 24 for 2 min according to the instructions supplied with the unit. Alternatively, if using 1.5 ml microcentrifuge tubes, recover the DNA by centrifugation at full speed for 1 min in a microcentrifuge. 7. Alternatively, if using 1.5 ml microcentrifuge tubes, recover the plasmid DNA by centrifugation at full speed for 1 minute in a microcentrifuge. Note: The recovered elution volume will be 40 60% of the applied volume for the MicroPlex 24 spin method of sample recovery. DNA recovery will be approximately 30% to 40% lower than that with the microfuge procedure. p18

19 Troubleshooting Problem: The DNA yield is low. Possible causes/solutions 1. The gel band was not fully dissolved: Incubate the gel band at 60 C for the full 15 min. Visually inspect the sample to make sure it is dissolved before proceeding. Make sure that the size of the gel band does not exceed 300 mg and that the proper volume of capture buffer is added. 2. The wash buffer was not completely removed before elution: Make sure that the GFX column is centrifuged for at least 30 s before the elution buffer is added. If humidity is high, increase the spin time to 1 min. Problem: The DNA sample floats out of the well when loading a gel. Possible causes/solutions 1. The wash buffer was not completely removed before elution: Make sure that the GFX column is centrifuged for at least 30 s before the elution buffer is added. If humidity is high, increase the spin time to 1 min. 2. Some of the final wash remained in the column below the frit and was collected in the final elution: The collection tube was not emptied after the initial sample was spun through the column. This caused the collection tube to overfill when the wash step was performed. Empty the collection tube as described in the procedure. If necessary, place the column back into the collection tube and spin briefly to remove any residual fluid. p19

20 Problem: DNA does not cut to completion with restriction enzymes/does not ligate. Possible causes/solutions 1. Some of the final wash remained in the column below the frit and was collected in the final elution: The collection tube was not emptied after the initial sample was spun through the column. This caused the collection tube to overfill when the wash step was performed. Empty the collection tube as described in the procedure. If necessary, place the column back into the collection tube and spin briefly to remove any residual fluid. If problems persist, please contact GE Healthcare Technical Service for assistance. p20

21 References 1. Vogelstein, B. and Gillespie, D., Proc. Natl. Acad. Sci. USA 76, 615 (1979). 2. Marko, M. A., Chipperfield, R. and Birnboim, H. C., Anal. Biochem. 121, 382 (1982). p21

22 Companion products available from GE Healthcare Product Product number Taq DNA Polymerase See GE Healthcare Catalog Ready-To-Go PCR Beads (100 reactions in 0.5 ml tubes) MicroPlex 24 See GE Healthcare Catalog MicroPlex 24 Vacuum See GE Healthcare Catalog MicroPlex 24 V-Bottom Collection Trays See GE Healthcare Catalog MicroPlex 24 Vacuum Gaskets See GE Healthcare Catalog MicroPlex 24 Vacuum Accessory Kit See GE Healthcare Catalog GenomicPrep Blood DNA Isolation Kit GenomicPrep Cells and Tissue DNA Isolation Kit DNA Polymerization Mix Base-Pair Ladder Base-Pair Ladder KiloBase DNA Marker A full range of sequencing kits and reagents can be found in the GE Healthcare catalog. p22

23 All goods and services are sold subject to the terms and conditions of sale of the company within the General Electric Company group which supplies them. A copy of these terms and conditions is available on request. * U.S. Patent No. 5,603,899 has been issued to Pharmacia Biosciences for multiple column chromatography. The Polymerase Chain Reaction (PCR) is covered by patents owned by Roche Molecular Systems and F Hoffmann-La Roche Ltd. A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers such as GE Healthcare and affiliates when used in conjuction with an authorized thermal cycler. Printed in the United States. p23

24 Trademarks AGE Healthcare are trademarks of General Electric Company. GFX, MicroSpin, MicroPlex, Ready-To-Go and GenomicPrep are trademarks of GE Healthcare Bio-Sciences Ltd. GE Healthcare UK Ltd GE Healthcare Place, Little Chalfont, Buckinghamshire, England HP7 9NA tel fax GE Healthcare Bio-Sciences AB SE Uppsala, Sweden tel 46 (0) fax 46 (0) GE Healthcare Bio-Sciences Corp 800 Centennial Avenue, PO Box 1327, Piscataway, NJ USA tel fax or GE Healthcare Europe GmbH Munzinger Strasse 9, D-79111, Freiberg, Germany tel fax p24