216
`
`NOTES & TIPS
`
`High-Resolution Preparative-Scale Purification
`of RNA Using the Prep Cell’
`
`Peristaltic
`Pump
`
`UV
`monitor
`
`Chart Recorder
`Digitizer
`
`Tu H. Nguyen,’ Lynette A. Cunningham,”
`Kendra M. Hammond, and Yi Lu®
`Department of Chemistry, University of Illinois,
`Urbana, Illinois 61801
`
`Received December 2, 1998
`
`Milligram-scale purification of RNA with high reso-
`lution is required for spectroscopic and X-ray crystal-
`lographic characterizations, as well as for clinicaltri-
`als. The presence of many conformations of the same
`RNA sequence makesit particularly difficult to purify
`RNA using column chromatography. Therefore, dena-
`turing gel electrophoresis is commonly used for RNA
`purification (1, 2). However, most gel electrophoresis
`methodssuffer low capacity, requiring 8-15 gels for a
`typical spectroscopic or X-ray structural experiment
`(3). We previously reported a method for automated
`large-scale purification of an RNA ribozyme from other
`transcription components using the Bio-Rad Prep Cell
`(4). Here we test a new model of the Prep Cell (Fig. 1)
`whichis three times longer than the original Model 491
`apparatus andalso investigate the sample recovery of
`the Model 491 apparatus. We found that the new ap-
`paratus offers significant improvement in resolution
`and loading capacity, and that the sample recovery of
`~90% for Model 491 apparatus is better than that
`typically obtained from either electroelution or crush-
`and-soak methods. This method can be easily adapted
`to large-scale purification of other nucleic acids.
`
`Materials and Methods
`
`A 100-ml-scale transcription was performed as pre-
`viously described to obtain milligram quantities of the
`34-mer 5’-GGCGACCGUGAUGAGGCCGAAAGGC-
`CGAAACAUU-3’ (4). The crude transcript was etha-
`nol-precipitated and reconstituted in 5 ml of 1.5x TBE‘
`(135 mM Tris—borate, 3 mM EDTA). The sample was
`then concentrated using Centricon-10 units (Amicon,
`Beverly, MA) to a final volume of 1-2 ml. Duringthis
`concentration procedure, the centricon was washed
`several times with 1.5<x TBEto partially remove un-
`
`‘This research was supported by the NIH FIRST Award
`(GM53706) and the Donors of the Petroleum Research Fund, admin-
`istered by the American Chemical Society.
`* Indicates an equal contribution to this work.
`* To whom all correspondence should be addressed at Department
`of Chemistry, University of Illinois, Box 8-6 Chemical and Life Sci-
`ences Laboratory, Urbana, IL 61801. E-mail: yi-lu@uiuc.edu.
`* Abbreviation used: TBE, Tris—borate-EDTA.
`
`Analytical Biochemistry 269, 216-218 (1999)
`Article ID abio.1999.4030
`0003-2697/99 $30.00
`Copyright © 1999 by Academic Press
`All rights of reproduction in any form reserved.
`
`(-)
`
`Fraction
`5, Collector
`
`Migrating RNA
`
`Gelsurface
`
`Beta Prep Cell
`
`FIG. 1. Schematic diagram of the modified Prep Cell. This modelis
`identical to the Model 491 except the lower chamber hasbeen elon-
`gated to accommodatea longergel tube. The RNAis loaded onto the
`cylindrical PAGE gel where it migrates down the gel toward the
`positive electrode (see the arrows), just as in traditional electro-
`phoresis. The separated RNA species are then pulled through the
`capillary tubing located in the center of the cooling core by a peri-
`staltic pump into a UV monitor andfinally into a fraction collector.
`A cellulose membrane with a molecular weight cutoff below that of
`the RNAis placed at the bottom of the Prep Cell gel to prevent the
`purified RNA from escaping the system while still allowing passage
`of conducting ions. A digitizer was also addedto allow collection of a
`computerized chromatogram in addition to the hardcopy chromato-
`gram generated bythestrip chart recorder.
`
`incorporated NTPs and thus increase the solubility of
`the transcript.
`Purification of the RNA transcripts was performed
`using the Model 491 Prep Cell and prototype Prep Cell
`along with equipment donated by Bio-Rad Laborato-
`ries, including the Model 1327 Econo-Recorder, EM-1
`Econo UV Monitor, Model 2128 Fraction Collector,
`EP-1 Econo Pump, and the Powerpac 1000 powersup-
`ply. In addition to the chart recorder, the data were
`simultaneously digitized using a ComputerBoard
`DAS-08 digitizer. A 20% acrylamide/8 M urea gel (120
`ml for a 13-cm gel, 260 ml for a 30-cm gel) was pre-
`pared in the 37-mm i.d. large Prep Cell gel tube and
`allowed to polymerize for 3 h. During the polymeriza-
`tion process, the gel was cooled using the recirculation
`pump connected to the cooling core and a 1-liter beaker
`of ice-water. To prevent crystallization of the urea dur-
`ing the cooling process, room-temperature water was
`passed through the pump until polymerization began
`and the gel started to get warm,at which point ice was
`added to the beaker. For each run, 130 yl of crude
`transcript was combined with an equal volumeoffor-
`mamide, heat denatured, and loaded onto the Prep Cell
`gel. We found that increasing the power to 15 W al-
`lowed for a shorter running time without loss in reso-
`lution. Therefore, the following separation conditions
`were used: 20% denaturing polyacrylamide, 15 W con-
`
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`

`NOTES & TIPS
`
`217
`
`stant power, 1.5 TBE running buffer, 1 ml/min elu-
`tion rate, and 8 ml/fraction.
`
`Results and Discussion
`
`Percentage sample recovery. To determine how
`much RNA can be recovered from the Prep Cell gel,
`duplicate runs were performed using the Model 491
`Prep Cell. For each run, 2-3 mg of purified 34-mer
`hammerhead ribozyme (quantified by UV absorption
`USING €2¢0nm = 285,483 M~' cm™') was loaded onto a
`freshly prepared 13-cm, 20% PAGEPrepCell gel. After
`completion of the run, the fractions containing RNA
`were combined, concentrated, desalted, and quantified
`by UV absorption. Of the 3.1 mg loadedin thefirst run
`andthe 2 mg loadedin the second,2.8 and 1.8 mg were
`recovered, respectively. The corresponding average re-
`covery is 90%. This recovery is better than that typi-
`cally obtained from both conventional electroelution
`(70—80%) (5) and crush-and-soak methods (60-80%)
`(6). The improved recovery is attributed to less post-
`electrophoresis manipulation.
`Effect ofgel height. The maximalgel height for the
`commercially available Model 491 Prep Cell is 13 cm,
`comparedto 40 cm for most preparative PAGEgels. To
`investigate whether a longer gel would offer better
`resolution, we used a beta Prep Cell model with a
`maximalheight of 40 cm, supplied by Bio-Rad Labora-
`tories (Hercules, CA). Figure 2A shows a comparison of
`chromatograms from purification runs using 13- and
`30-cm Prep Cell gels, each loaded with an RNA tran-
`script containing 0.8 mg of 34- and 35-mer hammer-
`head ribozyme (as determined by quantifying these
`bands on an analytical PAGE gel prior to Prep-Cell
`purification). The operating conditions were constant
`for each run. As expected, better resolution is achieved
`with the 30-cm gel. Individual peaks corresponding to
`n-—1,n,n+1,and n+ 2 transcripts are visible. To
`obtain comparable resolution on the 13-cm gel, less
`than half as much crude transcript can be loaded (4).
`The Prep Cell can be used to purify even larger
`quantities of RNA. An RNAtranscript containing ~7
`mg of 34- and 35-mer hammerhead ribozymes was
`loaded onto a 30-cm Prep Cell gel. Although resolved
`peaks corresponding to the 34- and 35-mer are not
`visible in the chromatogram,individual 8-ml fractions
`contain pure 34-mer RNA (Fig. 2B). Depending on the
`purity requirement of the particular application, those
`fractions containing less pure RNA can be pooled and
`repurified using the same Prep Cell gel to recover RNA
`of higher purity.
`In summary, the Prep Cell method provides high-
`resolution preparative purification of RNA with high
`percentage sample recovery. In addition, the longer
`apparatus has the advantageof allowing resolution of
`nfrom n+ 1 transcripts even whenseveral milligrams
`
`
`0.05 > -
`——
`
`0.04 +
`
`0.03 |
`
`0.02 +
`
`0.01 |
`
`Absorbanceat260nm
`
`30 cm
`
`0.00 + — a ~~ -
`
`
`0
`100
`200
`300
`400
`
`
`
`|
`
`|
`
`4
`
`Relative time (minutes)
`
`.Se eee es aRzee
`
`B
`
`(A) Overlaid chromatograms from purifications using 13-
`FIG. 2.
`and 30-cm PrepCell gels. Because the elution timediffers for the two
`gel lengths, the origin of the time axis shown correspondsto a point
`just before peak elution and does not correspondto the start of each
`run. The origin correspondsto 15.5 h (930 min) and 47.5 h (2850 min)
`for the 13- and 30-cm runs,respectively. (B) Analytical PAGEgel of
`Prep Cell peak fractions from a 30-cm Prep Cell run in which an RNA
`transcript containing 7 mg of 34- and 35-mer hammerhead ribozyme
`wasloaded. Aliquots of every other fraction from the RNA peak were
`loaded onto a 19 cm X 29 cm X 0.7 mm gel and stained with ethidium
`bromide.
`
`is loaded. This method requires a significantly lower
`amount of acrylamide gel solution (~260 mlfor the 30
`cm Prep Cell) than a typical preparative PAGE gel
`(~720 ml for a 40 X 60 X 0.3 cm gel), needs no attended
`operation once the sample is loaded, and requires min-
`imal postelectrophoresis manipulation. Unlike the
`crush-and-soak(6), electroelution (5), or ultracentrifu-
`gation methods, the gel is not destroyed and can be
`used up to three times without significant loss of res-
`olution. These features make the Prep Cell method a
`viable alternative to traditional purification methods,
`especially for laboratories that need large quantities of
`RNA on a routine basis. This method has also been
`adapted to high-resolution purification of milligram
`quantities of other nucleic acids, such as phosphoro-
`thioate DNA and RNAin ourlaboratory.
`
`Acknowledgments. We thank J. J. Dunn and A.H. Rosenbergfor
`plasmid pAR1219 containing the T7 RNA polymerase gene, Pascale
`Legault and Arthur Pardi for the transcription protocol, Eric Val-
`lenderfor technical assistance, and Mary AnnIreland, Lauri Heerdt,
`and Linda Castle at Bio-Rad Laboratories for generously providing
`materials, equipment, and technical support. Y.L.
`is a Sloan Re-
`search Fellow of the Alfred P. Sloan Foundation, a Beckman Young
`Investigator of the Arnold and Mabel Beckman Foundation, and a
`Cottrell Scholar of the Research Corporation.
`
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`

`218
`
`REFERENCES
`
`NOTES & TIPS
`
`TABLE 1
`
`Efficiency of DNA Recovery from Agarose Using either the
`Original Wizard Direct Elution Method (7) or the Presented
`Improved Wizard Plus SV Salt/Freeze Method
`
`Fragmentsize (kb) Original method (%)
`
`Improved method (%)
`
`23
`9
`6.5
`
`33
`45
`40
`
`75
`85
`80
`
`1. Milligan, J. F., Groebe, D. R., Witherell, G. W., and Uhlenbeck,
`O. C. (1987) Nucleic Acids Res. 15, 8783-8798.
`2. Heus, H. A., Uhlenbeck, O. C., and Pardi, A. (1990) Nucleic Acids
`Res. 18, 1103-1108.
`3. Heus, H. A., and Pardi, A. (1991) J. Mol. Biol. 217, 113-124.
`4. Cunningham, L., Kittikamron, K., and Lu, Y. (1996) Nucleic
`Acids Res. 24, 3647-3648.
`5. Zassenhaus,H. P., Butow, R. A., and Hannon,Y. P. (1982) Anal.
`Biochem. 125, 125-130.
`6. Grierson, D. (1982) in Gel Electrophoresis of Nucleic Acids: A
`Practical Approach (Rickwood, D., and Hames,B. D., Eds.), p. 11.
`IRL Press, Oxford.
`
`An Improved Methodfor the Purification
`of Large DNA Fragments from Agarose
`Gels Using Wizard Plus SV Columns
`
`Daniel Tillett and Brett A. Neilan’'
`
`School of Microbiology and Immunology, University
`of New South Wales, Sydney, 2052, Australia
`
`Received December8, 1998
`
`agarose gel in 1X Tris—acetate-EDTA buffer (1). The
`gel was stained with ethidium bromide andthe 23-, 9-,
`and 6.5-kb bands were excised from both lanes under
`UV transillumination. Individual DNA fragments were
`eluted using either of the following two protocols:
`1. The original Wizard DNA gel elution protocol of
`Wolff and Hull
`(7). Briefly, gel slices were placed
`in individual standard Wizard columnsheld in 1.5-ml
`Eppendorf tubes and the DNA was eluted by centrifuga-
`tion at 14,000g for 12 min. DNA was precipitated by the
`addition of 0.1 vol of 3 M sodium acetate and 1 volof iso-
`propanol, followed by centrifugation at 14,000g for 10 min.
`The Wizard Plus SV column with salt equilibration
`and freezing protocol. Gel slices were placed within
`individual 2-ml Eppendorf tubes containing 1 mlofsalt
`Theisolation of DNA fragments from agarosegels is
`buffer (300 mM sodium acetate, 50 mM Tris—HCl, 1 mM
`an integral step of many molecular biological protocols.
`EDTA, pH 8.3). The gel slices were allowed to equilibrate
`Of the numeroustechniques developed to recover DNA
`for 30 min at room temperature before the gel slice was
`fragments from agarose gels (1), the direct elution of
`transferred, with minimal buffer, to individual Wizard
`DNA from the agarose matrix by centrifugation
`Plus SV miniprep DNA purification columns held in
`throughafilter is the simplest. Different filters have
`1.5-ml Eppendorf tubes. After freezing the gel slices for
`been used including cotton-filled pipet tips (2), glass
`10 min at —70°C,the excised DNA fragment was eluted
`wool (3), blotting paper (4), paper slurry (5), commer-
`by centrifugation at 14,000g for 12 min. DNA was pre-
`cial barrier pipet tips (6), and Wizard minicolumns(7).
`cipitated by the addition of 1 vol of isopropanol followed
`While the Wizard minicolumnis a convenient and re-
`by centrifugation at 14,000g for 10 min.
`producible means to directly elute DNA from agarose
`DNAsamples were resuspended in 10 pl of TE (10
`gels, it suffers from two limitationsasa filter. First, the
`mM Tris-HCl, pH 7.4; 1 mM EDTA,pH 8.0) and the
`standard Wizard columnsare of low capacity and its
`DNArecovery quantified (Table 1) using the Fluro-S
`small opening can maketheinsertion ofthe agarose gel
`Multilmager (Bio-Rad, Hercules, CA) after electro-
`slice awkward. Second, the DNAyield is often poor,
`phoresis on a 0.7% agarose gel with 30 ng of A—HindIII
`particularly with large DNA fragments. This problem
`DNA marker (Fig. 1).
`is, however, not confined to the use of Wizard colums
`The Wizard Plus SV column, in combination with a
`because low yields of large DNA fragments have been
`salt preequilibration and freeze step, providesa reli-
`observed with otherfilter systems (2-6).
`able method for the direct elution of large DNA frag-
`Wedescribe the use of Wizard Plus SV miniprep
`ments from agarose gels. Their large capacity and
`DNApurification columns (Promega, Madison, WI) for
`opening make them particularly convenient when ex-
`the rapid isolation of DNA fragments from agarose
`cising large gel volumes. The addition of a salt buffer
`gels. The yield of large DNA fragmentsis improved by
`preequilibration and freezing step provides for a sig-
`preequilibrating the gel slices in a neutral salt buffer
`nificant increased recovery of large DNA fragments
`and freezing before centrifugation (8).
`(Table 1). We have isolated DNA fragments using this
`Two 30-ng samples of A—HindIII-digested DNA
`technique from 0.4 to 3% agarose. Finally, DNA puri-
`marker were electrophoresed in parallel on a 0.7%
`fied using this method has proven suitable for a range
`of molecular biological procedures, including plasmid
`preparation, DNA ligations, DNA sequencing, PCR
`amplification, and restriction enzyme digestions (9).
`
`‘To whom correspondence and reprint requests should be ad-
`dressed. Fax: 61 2 9385 1591. E-mail: b.neilan@unsw.edu.au.
`
`Analytical Biochemistry 269, 218-219 (1999)
`Article ID abio.1999.4006
`0003-2697/99 $30.00
`Copyright © 1999 by Academic Press
`All rights of reproduction in any form reserved.
`
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