Transcript
QuickPrep™ Micro mRNA Purification Kit QuickPrep™ Micro mRNA Purification Kit* is designed for the direct isolation of polyadenylated RNA from small amounts of eukaryotic cells or tissues, bypassing the need for intermediate purification of total RNA. Purifications can be performed using a microcentrifuge or in conjunction with MicroPlex™ 24 Vacuum. Using MicroPlex 24❖, up to 24 samples can be processed in as little as 1 hour. Using either method, mRNA can be purified from one cell or from as many as 1 x 107 cells (~100 mg of tissue). Each purification allows the isolation of up to 6 µg of RNA. Purity will vary depending on the sample; typically 90% or more of the isolated RNA will be polyadenylated. The mRNA is suitable for numerous end uses. Sufficient reagents are provided for a total of 24 separate mRNA purifications.
XY-035-00-20 Rev. 10
CONTENTS Components Overview Protocol: Introduction Procedures: A. Preparation of Oligo(dT)-Cellulose B. Extraction of Sample C. Isolation of mRNA D. Quantitation and Precipitation of mRNA Appendices: 1. Additional Materials 2. Streamlined Washing Steps 3. Protocol for Use With MicroPlex 24 Vacuum Function Testing and Storage References Ordering Information Companion Products
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COMPONENTS Oligo(dT)-Cellulose: Oligo(dT)-cellulose (25 mg/ml) suspended in a buffer containing 0.15% Kathon™ CG/ICP Biocide. Extraction Buffer: A buffered aqueous solution containing guanidinium thiocyanate and N-lauroyl sarcosine.
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High-Salt Buffer: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 M NaCl. Low-Salt Buffer:
10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 M NaCl. Elution Buffer: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. Glycogen Solution: Glycogen at 5-10 mg/ml in DEPC-treated water. K Acetate Solution: 2.5 M potassium acetate (pH 5.0). MicroSpin™ Columns: 25 polypropylene minicolumns, each fitted with a frit. Caution: Extraction Buffer and K Acetate Solution are irritants and should be handled with care. Additional reagents and the equipment required are listed in Appendix 1, page 18.
Note: Before using a column, snap off the bottom closure. This portion can then be inverted to plug the column.
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OVERVIEW QuickPrep Micro mRNA Purification Kit combines the disruptive and protective properties of guanidinium thiocyanate (GTC, references 1 and 2) with the speed and selectivity of oligo(dT)-cellulose chromatography in a spun-column format pioneered by GE Healthcare (3). The kit is designed for the rapid isolation of mRNA from small amounts of eukaryotic cells or tissue without the need for intermediate purification of total RNA. Starting with cultured cells, mRNA can be purified in just 15 minutes; purification of mRNA from tissue samples will take several minutes longer, due to the need for homogenization of the tissue. The kit can be used to isolate mRNA from as little as one cell or as many as 1 x 107 cells (~100 mg of tissue). The protocol for mRNA purification using a microcentrifuge is given in Procedures A-D and is outlined in the flow chart opposite. The protocol for using this kit in conjunction with MicroPlex 24 Vacuum is provided in Appendix 3, page 20. The following summarizes the steps involved when using the standard microcentrifuge-based protocol. Briefly, the tissue or cells are extracted in a buffered solution containing a high concentration of GTC, ensuring the rapid inactivation of endogenous RNases. The extract is then diluted three-fold with Elution Buffer, reducing the GTC concentration to a carefully selected level - low enough to allow efficient hydrogen-bonding between poly(A) tracts on the mRNA molecules and oligo(dT) attached to cellulose, but high enough to maintain complete inhibition of RNases. The three-fold dilution causes a number of proteins to precipitate, giving an initial purification.
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The extract is then clarified by a short centrifugation, and the supernatant transferred to a microcentrifuge tube containing Oligo(dT)Cellulose. After several minutes, during which the poly(A)+ RNA binds to the Oligo(dT)-Cellulose, the tube is centrifuged at high speed for 10 seconds, and the supernatant pipetted or aspirated off the pelleted Oligo(dT)-Cellulose. The pelleted material is then washed sequentially with 1-ml aliquots of High-Salt Buffer and Low-Salt Buffer, each wash being accomplished by a process of resuspension and brief (10-second) re-centrifugation (referred to hereafter as batch-washing). After the last batch-wash with Low-Salt Buffer, the pelleted Oligo(dT)Cellulose is brought up in a small volume of Low-Salt Buffer. The slurry is transferred to a MicroSpin Column placed within a microcentrifuge tube, and the column is washed with three 0.5 ml aliquots of Low-Salt Buffer, with a 5-second centrifugation between each addition of buffer. (Alternatively, a streamlined washing protocol in Appendix 2 may be used which yields slightly less pure mRNA but speeds up the isolation process by eliminating some of the washing steps.) Finally, the polyadenylated material is eluted with one or two applications of 0.2 ml of pre-warmed Elution Buffer. Each purification will yield a maximum of 6 µg of RNA. Purity will vary depending on the sample, with at least 50% and typically 90% or more of the isolated RNA being polyadenylated. The mRNA isolated with the kit is essentially free of DNA and protein contamination.
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mRNA purified with the kit has been used directly in first-strand cDNA synthesis prior to PCR† without dilution or concentration. Note, however, that the maximum concentration of mRNA likely to be obtained is 15 µg/ml, which may be insufficient for some end uses. For Northern analysis, standard cDNA synthesis and in vitro translation, we recommend that the RNA be concentrated prior to use. The kit contains both potassium acetate and glycogen for precipitation of the mRNA when required. † The PCR process is covered by U.S. Patent Nos. 4,683,195 and 4,683,202 owned by HoffmannLa Roche Inc. Use of the PCR process requires a license.
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PROTOCOL Below are several points which should be reviewed prior to beginning the procedures.
RNase-Free Conditions and Safety Precautions The most important consideration in any isolation of RNA is protection of the sample from contamination with ribonucleases (RNase). Because the reagents are packaged as “stock solutions” which will need to be drawn from on separate occasions, we recommend that the required amount of each reagent needed for a particular purification be transferred to sterile tubes before beginning the isolation procedure. This will help avoid possible contamination of the stock reagents. Any other plastic- or glassware which may come into contact with the sample should be autoclaved or otherwise treated to prevent RNase contamination (4). Fresh gloves should be worn during the purification, both to protect you from contact with solutions and to protect the RNA from nucleases present on your skin. Protective eyewear should be worn at all times.
Centrifugation and Microcentrifuge Tubes A microcentrifuge should be used which can generate a centrifugal force between 5,000 x g and 16,000 x g. The method was developed using an Eppendorf Model 5415; at full speed, it provides a g-force of 16,000. Standard 1.5 ml and 2.0 ml microcentrifuge tubes have been used in the development of QuickPrep Micro mRNA Purification Kit. We have found it convenient to use microcentrifuge tubes with attached caps for the initial binding and batch-washing procedures. Centrifugation time 8
during each of these washes is not critical. However, the minimum centrifugation time should be 10 seconds. We have found that a 10second pulse at 16,000 x g is sufficient to pellet the Oligo(dT)-Cellulose. Once the resin is poured into a MicroSpin Column, the centrifugation times should be consistent (5 seconds each). This is most easily monitored using a digital timer. Use of 2 ml microcentrifuge tubes decreases the number of times the collection tube must be removed and emptied. For the final elution step, we prefer to switch to screw-top microcentrifuge tubes.
Other Points The tubes should always be counterbalanced during centrifugation; this is especially critical during sample elution. Ideally, perform multiples of two purifications simultaneously for consistent balancing. Resin may be inadvertently removed during buffer aspiration. If the amount is minimal, it should not affect the purification. If a large amount of resin is accidentally removed during batch-washing, the aspirated liquid should be returned to the tube and the centrifugation and buffer removal repeated. Occasionally, a small amount of resin may leak through the frit of the MicroSpin Column. This should not affect the success of the mRNA purification. The capacity of the QuickPrep system is finite (see Overview, page 4). Overloading the system will result in a decrease in the purity of the mRNA isolated (as measured by the A260/A280 ratio). In the text which follows, materials provided as components of the kit appear in boldface type.
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Procedure A: Preparation of Oligo(dT)-Cellulose Preliminaries • Approximately 20-30 minutes before the tissue or cell sample will be ready for extraction, remove the kit from storage at 4°C and place it at room temperature. Remove the Extraction Buffer and place it at 37°C until all crystalline material is dissolved. Cool to room temperature. -
Note: If the crystalline material persists, place the bottle at 55ºC and shake occasionally. If it is difficult to get the final crystals into solution, simply allow the crystalline material to settle and pipet the solution away from the crystals. This will not result in any deleterious effects on buffer performance.
• Prepare cuvettes for A260 determination (see Appendix 1, page 18).
Preparation for Batch-Washes The Oligo(dT)-Cellulose has been extensively washed and is ready for immediate use in QuickPrep Micro mRNA Purification Kit. • Gently swirl the Oligo(dT)-Cellulose slurry to obtain a uniform suspension. • Immediately pipette 1 ml aliquots of Oligo(dT)-Cellulose into individual microcentrifuge tubes for each purification to be performed. • Cap the tubes and proceed to Procedure B.
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Procedure B: Extraction of Sample During extraction, disrupted cells may appear to “clump together”, or the homogenate may become quite viscous. Neither of these phenomena will affect the outcome of the extraction. Continue the procedure uninterrupted. If working with cultured cells or blood, proceed to page 12. Otherwise, proceed as described below.
For Extraction of Tissue (up to 0.1 g) The optimum method for preparation of tissue samples depends on tissue type. Below are various suggestions for tissue preparation. Ideally, you should choose your method of sample preparation based on past experience with the tissue of interest. We have included a slight excess of Extraction Buffer and Elution Buffer to aid in the determination of optimal extraction procedures. We have found that there is no need to freeze soft tissue prior to extraction. Tissue samples will need to be homogenized as the first step in mRNA extraction. This can typically be achieved using a homogenizer with a small capacity (7 ml). Many such homogenizers come with two pestles, a larger, tight-fitting one (referred to below as the “A pestle”) and a smaller, loose-fitting one (“B pestle”). Homogenization of the tissue can be performed using either 0.4 ml or 0.6 ml of Extraction Buffer. For tissue that is difficult to homogenize, we recommend the larger volume. With this larger volume, however, 2 ml microcentrifuge tubes are required for both the extraction and the binding steps. In the text below, the volumes in parentheses should be used if the extraction was performed by adding 0.6 ml of Extraction Buffer. If only one volume is given, it pertains to all samples, regardless of starting volume. • Place tissue in either a manual or a mechanical homogenizer and add 0.4 ml (or, for some samples, 0.6 ml) of Extraction Buffer for each 11
100 mg or less of material. • Homogenize the tissue until it is a uniform suspension. The tissue should be well homogenized in a 7 ml homogenizer using 10-20 strokes with the B (smaller) pestle followed by 10 strokes with the A (larger) pestle. • Dilute the sample by adding 0.8 ml (or, for some samples, 1.2 ml) of Elution Buffer (not Extraction Buffer!) to the extract and mix thoroughly. Homogenize briefly, then transfer the homogenate to a sterile tube. • Place 0.5 ml of Elution Buffer (per purification) at 65°C until needed in Procedure C.
For Extraction from Cells (up to 1 x 107 cells) • If dealing with tissue-culture cells, free adherent cells from anchorage by standard methodology and suspend them in a small volume of isotonic buffer. If working with blood samples, the kit can accommodate ~25 µl of whole blood. Blood samples can be used directly; there is no need to pellet them prior to extraction. Count the cells using a hemocytometer (5). • Place an aliquot of cell suspension containing between one and 1 x 107 cells in a microcentrifuge tube and pellet by centrifugation. Remove the supernatant. • Add 0.4 ml of Extraction Buffer to the pelleted cells. Vortex until a homogeneous suspension is achieved. • Dilute the sample by adding 0.8 ml of Elution Buffer (not Extraction Buffer!) and mix using a vortex mixer. • Place 0.5 ml of Elution Buffer (per purification) at 65°C until needed in Procedure C. 12
Procedure C: Isolation of mRNA Binding Step If MicroPlex 24 Vacuum is to used for "Washing Steps" (see page 14), assemble the MicroPlex 24 Vacuum according to the instructions supplied with the unit before proceeding. • Prepare a “cleared cellular homogenate” by centrifuging each extracted sample for 1 minute at top speed (between 5,000 x g and 16,000 x g). The tube containing Oligo(dT)-Cellulose (prepared in Procedure A, page 10) should also be centrifuged for 1 minute; this can be done in parallel with the centrifugation of the sample. • Remove the buffer from the Oligo(dT)-Cellulose pellet by aspiration or by using a 1 ml pipetting device. • Place 1 ml (or, for the larger sample volumes, 1.5 ml) of the cleared cellular homogenate on top of the pellet of Oligo(dT)-Cellulose. • Close the tube and invert to resuspend the Oligo(dT)-Cellulose. The Oligo(dT)-Cellulose may form small clumps, but this will not affect the procedure and should be ignored. • Gently mix for 3 minutes by inverting the tube manually or by placing it on a rocking table or similar device. • Place the sample in the microcentrifuge and centrifuge at a maximum of 16,000 x g for 10 seconds. • Remove the tube from the centrifuge, open the lid, and remove the supernatant by aspiration or pipetting. 13
Washing Steps The following protocol is designed for isolating mRNA that is essentially free of contaminating proteins, nucleic acids and carbohydrates. A streamlined washing procedure (Appendix 2, page 19) may be substituted for the following protocol in situations where highly purified mRNA is not required. See Appendix 3, page 20 for the protocol for use with MicroPlex 24 Vacuum. • Add 1 ml of High-Salt Buffer. Close the lid of the tube and resuspend the Oligo(dT)-Cellulose. Place the tube in the centrifuge and spin for 10 seconds. Remove the tube, open the lid, and remove the supernatant by aspiration or pipetting. • Repeat the wash using High-Salt Buffer four more times, exactly as described in the step above, for a total of five washes. • Add 1 ml of Low-Salt Buffer to the Oligo(dT)-Cellulose pellet and close the lid. Resuspend the resin by inversion. You may need to tap the bottom of the tube. Place the tube in the microcentrifuge and pellet the resin by centrifugation for 10 seconds. Open the tube and remove the supernatant by aspiration or pipetting. • Repeat the wash using Low-Salt Buffer one more time, exactly as described in the step above, for a total of two washes. • Resuspend the resin in 0.3 ml of Low-Salt Buffer and transfer the slurry to a MicroSpinColumn, placed in a microcentrifuge tube. • Place the column in the microcentrifuge and spin at full speed for 5 seconds. 14
• Remove the column from the microcentrifuge and discard the effluent in the collection tube. Replace the column in the tube and add 0.5 ml of Low-Salt Buffer. Avoid disturbing the cellulose bed. Centrifuge for 5 seconds at full speed. • Repeat the step above an additional two times, emptying the collection tube between each step if necessary, for a total of three washes.
Elution Step • Remove the column and place it in a sterile microcentrifuge tube, preferably screw-cap. • Place this tube in the microcentrifuge and add 0.2 ml of prewarmed Elution Buffer (prepared in Procedure B) to the top of the resin bed. • Centrifuge at top speed for 5 seconds. Do not discard the eluate – it contains your mRNA! -
Note: The 0.2 ml eluate will contain approximately 80-90% of the recoverable mRNA. The majority of the remaining mRNA can be captured with a second elution step (see below). A second elution step will, however, result in a more dilute mRNA sample which may need to be precipitated in order to concentrate the mRNA prior to end use (see Procedure D).
• Add a second (optional, see note above) 0.2 ml aliquot of warm Elution Buffer to the top of the resin bed and centrifuge as described above. • Remove the column and place the tube containing the eluted mRNA on ice for quantitation, precipitation, or direct end use.
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Procedure D: Quantitation and Precipitation of mRNA Quantitation The concentration of mRNA in the final eluate can be determined by spectrophotometry. The minimum absorbance which can be relied upon to be accurate is 0.05, equivalent to an RNA concentration of 2 µg/ml. We suggest that you determine the concentration of the mRNA using undiluted eluate, placing the sample in a pretreated cuvette (Procedure A and Appendix 1) and recovering it for end use. The concentration of mRNA can be determined using the following formula: [RNA] = A260 x 40 µg/ml. -
Note: It is possible that residual oligo(dT)-cellulose fines may interfere with accurate quantitation of the mRNA. To remove these fines from the sample of eluted mRNA, centrifuge at full speed in a microcentrifuge for 1 minute. Carefully pipet the supernatant into a clean microcentrifuge tube. Discard the original tube.
Precipitation The mRNA may not need to be precipitated if it is to be used for PCRrelated procedures. However, mRNA samples which are to be used for standard cDNA, Northern analysis, and translation often need to be concentrated by precipitation prior to end use, as follows: • Add 10 µl of Glycogen Solution and 40 µl of K Acetate Solution (1/10 volume) to the 400 µl of sample. Add 1 ml of 95% ethanol (chilled to -20°C) and place the sample at -20°C for a minimum of 30 minutes. 16
• Collect the precipitated mRNA by centrifugation in a microcentrifuge at 4°C for 5 minutes. If the RNA is not to be used immediately, store it in this precipitated state (under ethanol) at -80ºC. • Decant the supernatant and invert the tube over a clean paper towel. Gently tap the tube on the towel to facilitate the removal of excess liquid. • Redissolve the precipitated RNA in an appropriate volume of Elution Buffer or DEPC-treated water. To determine the “appropriate” volume, consider the RNA concentration desired, the concentration before precipitation ([RNA]), and the volume of the sample subjected to precipitation. Note, however, that the percentage of the RNA recovered after precipitation will depend on the total amount present. With 5 µg of RNA, for example, ~ 70% will be recovered. You may therefore wish to redissolve the pellet in a volume 25-50% smaller than would be required if all of the RNA were recovered.
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Appendix 1: Additional Materials As noted on page 8, all plastic- or glassware which comes into contact with the sample should be treated to prevent RNase contamination (5). • Mechanical or manual tissue homogenizer (optional). • 1.5 and/or 2 ml microcentrifuge tubes. • 1 ml pipettes, or pipetting device. • Microcentrifuge capable of generating a g-force between 5,000 and 16,000. • 95% ethanol at -20°C. • Spectrophotometer (optional). • 0.5 ml quartz glass cuvettes (optional): Treat by soaking in concentrated HCl/methanol (1:1) for 1 hour and then rinsing several times in DEPC-treated water (see below). • DEPC-treated water: Prepare a 0.1% solution of diethyl pyrocarbonate (DEPC) in distilled water, allow it to stand overnight at room temperature, then autoclave (see also reference 4).
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Appendix 2: Streamlined Washing Steps The following protocol may be substituted for the washing steps described on page 14. The streamlined protocol can be utilized when downstream uses do not require high-purity mRNA (e.g. RT-PCR)*. • Add 1 ml of High-Salt Buffer. Close the lid of the tube and resuspend the Oligo(dT)-Cellulose. Place the tube in the centrifuge and spin for 10 seconds. Remove the tube, open the lid, and remove the supernatant by aspiration or pipetting. • Repeat the wash using High-Salt Buffer one to two more times, exactly as described above, for a total of two to three washes. • Add 1 ml of Low-Salt Buffer to the Oligo(dT)-Cellulose pellet and close the lid. Resuspend the resin by inversion. You may need to tap the bottom of the tube. Pellet the resin by centrifugation for 10 seconds. Open the tube and remove the supernatant by aspiration or pipetting. • Repeat the wash using Low-Salt Buffer one to two more times, exactly as described in the step above, for a total of two to three washes (the total number of Low-Salt washes should equal the number of High-Salt washes performed). • Resuspend the resin in 0.3 ml of Low-Salt Buffer and transfer the slurry to a MicroSpinColumn, placed in a microcentrifuge tube. • Place the column in the microcentrifuge and spin at full speed for 5 seconds. Proceed to Elution Step, page 15. * While purity will vary depending on sample, as a general rule, two High-Salt washes will remove approximately 80-90% of contaminating proteins and nucleic acids. A third High-Salt wash can be used to achieve even greater purity. Five washes may be needed to completely remove carbohydrates.
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Appendix 3: Protocol for Use With MicroPlex 24 Vacuum • Add 1 ml of High-Salt Buffer to each sample. Close the lid of each tube and resuspend the Oligo(dT)-Cellulose. Place the tubes in a microcentrifuge and spin for 10 seconds. Remove the tubes, open the lids, and remove the supernatants by aspiration or pipetting. • Add 500 µl of High-Salt Buffer to each tube. Using a pipettor, resuspend each cellulose pellet. Transfer the slurry from each tube to an empty MicroSpin Column that has been placed in the prepared MicroPlex assembly (see instructions which accompany MicroPlex 24). -
Note: The vacuum should be turned off at this step and the stopcock should be closed (i.e. should be perpendicular to the direction of the tubing).
• Engage the vacuum by turning the stopcock slowly just to the point where the liquid begins to evacuate the columns. Leave the stopcock at this position until no liquid remains in the cellulose. Maintain the vacuum for 2-3 seconds more. Turn the stopcock to the off position. -
Note: It is not unusual for wells to evacuate at slightly different rates.
• Add 500 µl of High-Salt Buffer to the cellulose pellet. Apply the vacuum as described above. Repeat this step 0-2 times, depending on the desired purity of the mRNA. • Remove the V-bottom collection tray from the MicroPlex assembly. Place the MicroPlex manifold plus gasket on top of a new V-bottom collection tray. If the gasket has been dislodged from its proper position, reposition it while keeping the MicroPlex manifold upright. • Add 500 µl of Low-Salt Buffer to the cellulose pellet. Engage the vacuum by turning the stopcock slowly just to the point where the liquid begins to evacuate the columns. Leave the stopcock at this 20
position until no liquid remains in the cellulose. Maintain the vacuum for 2-3 seconds more. Turn the stopcock to the off position. • Repeat the wash using Low-Salt Buffer two more times, exactly as described in the step above, for a total of three low-salt washes. • Remove the V-bottom collection tray from the MicroPlex assembly. Place the MicroPlex manifold plus gasket on top of a new V-bottom collection tray. If the gasket has been dislodged from its proper position, reposition it while keeping the MicroPlex manifold upright. • Add 200 µl of pre-warmed Elution Buffer to the top of the cellulose pellet. Apply the vacuum as described previously. The V-bottom collection tray now contains the eluted mRNA. -
Note: The 0.2 ml eluate will contain approximately 80-90% of the recoverable mRNA. The majority of the remaining mRNA can be recovered with a second elution step (see below). A second elution step will, however, result in a more dilute mRNA sample which may need to be precipitated in order to concentrate the mRNA prior to end use (see Procedure D).
• Add a second (optional, see note above) 0.2 ml aliquot of warm Elution Buffer to the top of the resin bed and apply the vacuum as described previously. • At this point, the eluted mRNA is ready for quantitation, precipitation or direct use. The samples can be stored in the V-bottom collection tray covered with a plate sealer or may be transferred to microcentrifuge tubes. -
Note: If the samples will be quantified using a spectrophotometer, we recommend transferring the samples to microcentrifuge tubes and spinning at full speed in a microcentrifuge for 1 minute to remove the cellulose fines. Carefully pipet the supernatant into a clean microcentrifuge tube. Discard the original tube.
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FUNCTION TESTING Each lot of QuickPrep™ Micro mRNA Purification Kit is tested for its ability to extract young rabbit whole blood mRNA suitable for direct use in PCR and cDNA synthesis.
STORAGE Store at 4°C.
REFERENCES 1. 2. 3. 4.
Chirgwin, J. M. et al., Biochemistry 18, 5294 (1979). Pharmacia P-L Biochemicals, Analects 17.4 (1989). Pharmacia P-L Biochemicals, Analects 16.2 (1988). Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, second edition (1989). 5. Ausubel, F. M. et al., eds., Current Protocols in Molecular Biology 2, 11.5.1 (1989).
ORDERING INFORMATION QuickPrep™ Micro mRNA Purification Kit
24 purifications
27-9255-01
Companion Products For Cell Separation Percoll™
250 ml 1 liter 22
17-0891-02 17-0891-01
Ficoll-Paque™
6 x 100 ml 6 x 500 ml
17-0840-02 17-0840-03
Ficoll-Paque™ PLUS (Endotoxin-Tested)
6 x 100 ml
17-1440-02
For cDNA Synthesis and Cloning DNA Polymerization Mix 10 µmol of each dNTP Cohesive-End Adaptor, EcoR I/Not I 0.5 A260 units TimeSaver™ cDNA Synthesis Kit 5 cDNA syntheses Directional Cloning Toolbox 1 kit λ ExCell Not I/EcoR I/CIP 10 µg
27-2094-01 27-7791-01 27-9262-01 27-9274-01 27-5013-01
For Northern Hybridization T7QuickPrime™ Kit Dextran Sulfate Ficoll™ 400 VacuGene™ XL Blotting System Nitrocellulose Membrane GeneBind 45 Nylon Membrane For High Throughput Purification MicroPlex™ 24 Vacuum 23
40 reactions 100 g 500 g 100 g 500 g 1 unit 1 unit 1 unit
27-9252-01 17-0340-01 17-0340-02 17-0400-01 17-0400-02 80-1266-S1 80-1098-90 80-1247-87
1 system
27-3567-01
MicroPlex 24 V-Bottom Collection Tray MicroPlex 24 Vacuum Gaskets MicroPlex 24 Vacuum Accessory Kit MicroPlex 24
50 trays 20 gaskets 1 kit 1 set
27-3563-01 27-3568-01 27-3569-01 27-3564-01
QuickPrep™, MicroPlex™, MicroSpin™, Percoll™, Ficoll-Paque™, TimeSaver™, QuickPrime™, Ficoll™ and VacuGene™ are registered trademarks of GE Healthcare Bio-Sciences Ltd or its subsidiaries. GE Healthcare is a trademark of General Electric Company. Kathon™ CG, is a registered trademark of Rohm and Haas Company. *QuickPrep Micro mRNA Purification Kit is protected by GE Healthcare Bio-Sciences Ltd or its subsidiaries under U.S. Patent No. 5,459,253 and foreign equivalents. ❖MicroPlex 24 is pr otected by GE Healthcare Bio-Sciences Ltd or its subsidiaries under U.S. Patent No. 5,603,899 and foreign equivalents. All goods and services are sold subject to the terms and conditions of sale of the company within the General Electric Company group that supplies them. A copy of these terms and conditions of sale is available on request. Printed in the United States for GE Healthcare Bio-Sciences Inc. GE Healthcare UK Ltd. GE Healthcare Place, Little Chalfont, Buckinghamshire, England HP7 9NA. GE Healthcare Bio-Sciences AB . SE-751 84, Uppsala, Sweden. GE Healthcare Bio-Sciences Inc. 800 Centennial Avenue, PO Box 1327 Piscataway, NJ 08855, USA.
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